KR20080112413A - Drugs and uses - Google Patents

Abstract

The invention relates to methods to treat specified clinical disorders such as hyperglycemia, type 2 diabetes, arthritis and multiple sclerosis. The invention also provides methods to identify and characterize drugs, which are characterized in part by eliciting a variable biologic or therapeutic effect on a biomolecule at one time and relative normalization of the biomolecule at another time point. Compounds include 17alpha-ethynylandrost-5-ene-3beta,7beta, 17beta-triol or androst-5-ene-3beta,4beta, 16alpha, 17beta-tetrol, which can be used as reference standards to facilitate assessing and characterizing such candidate drugs. ® KIPO & WIPO 2009

Description

Drugs and their uses {DRUGS AND USES}

The present invention relates to compounds such as anstrost-5-ene-3β, 4β, 16α, 17β-tetrol, and methods of controlling such diseases, for controlling inflammation, metabolic diseases and other symptoms described herein. . The compounds that modulate inflammation are autoimmune such as type 2 diabetes, hyperglycemia, pulmonary inflammatory diseases such as cystic fibrosis, acute or chronic asthma, acute respiratory distress syndrome (ARDS) or chronic obstructive pulmonary disease (COPD) and multiple sclerosis. It can be used to treat, alleviate, prevent or slow the progression of symptoms such as disease, arthritis or lupus erythematosus.

Many factors contribute to the development and maintenance of various chronic autoimmune and inflammatory diseases. Usually the etiology of these diseases is unidentified. Tumor necrosis factor-α (TNF-α) is the first cytokine released by mononuclear phagocytes in response to a number of immune stimulants. When administered to animals or humans, fever, cardiovascular symptoms, bleeding, coagulation and acute conditions similar to those observed in acute infections and shock conditions are caused. If TNFα production is excessive or unregulated, it means several disease states. These include endotoxemia and / or toxic shock syndrome (e.g., Tracey et al, Nature 330:. 662-664 (1987) and Hinshaw et al, Circ Shock 30:.. 279-292 (1990), kakek cyano (cachexia ) (Eg, Dezube et al., Lancet , 335 (8690): 662 (1990)) and ARDS, wherein high concentrations of TNF α have been detected in lung inhalation fluid of ARDS patients (eg, Millar et al., Lancet 2 (8665): 712-714 (1989).

TNF α also appears to be involved in bone resorption diseases, including arthritis. When activated, leukocytes can cause bone resorption, which is the activity that TNFα can contribute to (eg Bertolini et al., Nature 319: 516-518 (1986) and Johnson et al., Endocrinology 124 (3): 1424-). 1427, (1989) TNFα also stimulates bone resorption and inhibits bone formation in vitro and in vivo through the inhibition of osteoblast function and stimulation of osteoclast formation and activation. Blocking TNFα using monoclonal anti-TNFα antibodies has been shown to affect rheumatoid arthritis (Elliot et al., Int. J. Pharmac . 17 (2): 141-145, 1995) and Crohn's disease. (von Dullemen et al., Gastroenterology , 109 (1): 129-135, 2005).

Nuclear factor-κB (NF-κB) molecules mediate inflammation in many clinical symptoms. Some therapeutic agents used to treat inflammation, such as dexamethasone, prednisone or hydrocortisol, are glucocorticoid receptor (GR) agonists that indirectly inhibit NF-κB by increasing the activity of glucocorticoids (eg, H. Harkonarson et al. , Am. J. Respir. Cell Mol. Biol . 25: 761-771, 2001). However, increased levels of natural GR agonists and pharmacological levels of synthetic GR agonists generally lead to significant immunosuppressive responses and undesirable toxicity, including bone mass loss or osteopenia (eg, TL Popper et al. , Anti-inflammatory agents: Anti-inflammatory steroids , RA.Scherer & MW Whitehouse, editors, Academic Press, New York, Chapter 9, volume 1, pages 245-294, 1974). Many of the unwanted toxicities associated with glucocorticoids are caused by the activation of GR. Therefore, identifying compounds that can inhibit NF-κB activity without inducing toxicity by activation of GR results in a class of therapeutic agents that can be used to treat related signs such as inflammation and pain, fever or fatigue. Can be.

In many chronic and acute symptoms such as ARDS, COPD and sepsis, undesirable or detrimental inflammatory reactions occur. Activated monocytes and neutrophils play an important role in mediating inflammation associated with pathogens. Of particular importance is activated neutrophils, which showed increased accumulation in the nucleus of NF-kB transcriptional regulators in the nucleus and increased production of proinflammatory cytokines. Neutrophils are also grades of toxic oxygen species that, when they occur, mediate at least in part the secretion of tumor necrosis factor-α (TNF-α) by activated monocytes, which may be necessary for the injury and damage of organs found in sepsis. It may be a circle.

Signaling associated with inflammation can occur by different pathways, which can increase the activity of NF-κB in infected cells. Activation of NF-κB by tumor necrosis factor-α (TNF-α) results from the binding of TNF-α to TNF-α receptors at the cell membrane followed by subsequent activation of signal transducers, including MAP kinases. When NF-κB is activated in the cytoplasm, it is transported to the nucleus and activates genes containing NF-κB response elements in their promoters. Activation of cytoplasmic NF-κB by bacterial Lipopolysaccharide (LPS) is characterized by intracellular binding of phosphatidylinositol-3-kinase, followed by LPS binding to toll-like receptor 4 at the cell surface. The signal transducer comes from being activated. TNF-α and LPS are both known to elicit potent inflammatory responses in cells in vivo and in vitro. Cells that respond to these pre-inflammatory signals include macrophages, monocytes and other types of immune cells.

Various T cell subsets appear to play an important role in the development of certain disease symptoms. An important role of separate populations of T cells, including regulatory and / or inhibitory T cells, in mediating various immune responses is described, for example, in E. Suri-Payer et al., J. Immunol. , 160 (3): 1212. -1218, 1998; J. Shimizu et al., J. Immunol ., 163 (10): 5211-5218, 1999; M. Itoh et al., J. Immunol ., 162 (9): 5317-5326, 1999 AM Miller et al., J. Immunol ., 177: 7398-7405, 2006). CD4 ⁺ CD25 ⁺ T cells may play an important role in inhibiting some immune responses.

The results of studies in animal models for some of these T cell subsets have been disclosed, for example, in US Pat. No. 6,593,511. For example, the role in the study of human autoimmune diseases was examined in the scid / scid CD4 ⁺ CD45Rb ^hi model. This animal model was used to study poorly regulated immune responses such as inflammatory symptoms and was used to validate experimental drugs and treatment protocols (eg K. Hong et al., J. Immunol ., 162: 7480-7491 , 1999; Powrie et al., J. Exp. Med ., 183 (6): 2669-2674, 1996).

The Foxpro3 gene is induced by thymic epithelial cells and may play an important role in inducing T cells to generate a CD4 ⁺ CD25 ⁺ or CD4 ⁺ CD25 ^high (Treg or regulator T cell) phenotype. CD25 surface antigen is the IL-2 receptor α chain. In some animal models of autoimmune diseases, deletion of the Foxpro3 gene is associated with the development of autoimmune diseases (eg, US Patent Application 2006/0111316). Recovery of the gene appears to alleviate abnormalities associated with autoimmunity. Various reagents or assay protocols for CD4 ⁺ CD25 ⁺ cells have been published (eg, H. Yagi et al., International Immunol ., 16 (11): 1643-1656, 2004; WR Godfrey et al., Blood , 105 (2) 750-758, 2005).

Insulin resistance in glucose intolerant subjects has long been known. Reaven et al ( American Journal of Medicine , 60 (1): 80-88, 1976) describe a technique for continuously injecting glucose and insulin to demonstrate the presence of insulin resistance in various groups of nonobese, nonketotic subjects ( Insulin / glucose clamp technique), and oral glucose tolerance test. These subjects ranged from borderline glucose tolerance to apparent fasting hyperglycemia. The diabetic group in these studies included insulin dependent (IDDM) subjects and insulin independent (NIDDM) subjects.

Sustained insulin resistance makes it easier to determine hyperinsulinemia, which can be measured by accurately measuring circulating plasma insulin levels in a subject's plasma. Hyperinsulinemia is the result of insulin injection compared to normal physiological release of hormones by the endocrine pancreas and as a result of insulin resistance in obese and / or diabetic (NIDDM) subjects and / or glucose intolerant subjects, or IDDM subjects May appear.

The relationship between obesity and large blood vessel ischemia (eg, atherosclerosis) and hyperinsulinemia has been published in experimental, clinical and epidemiological studies (Stout, Metabolism , 34: 7, 1985; Pyorala et. al, Diabetes / Metabolism Reviews , 3: 463, 1987). A statistically significant increase in plasma insulin concentration 1 and 2 hours after oral glucose administration is consistent with an increased risk of cardiovascular disease.

One model of human diabetes is db / db mice. db / db mice are described, for example, in D. Koya et al., The FASEB Journal, 14: 439-447, 2000; K. Kobayashi et al., Metabolism , 49 (1): 22-31, 2000; J. Berger et al., J. Biol. Chem ., 274 (10): 6718-6725, 1999. db / db mice carry mutations in genes encoding leptin receptors, and thus, over time, the functional pancreatic β-cell mass is impaired, resulting in phenotypes characterized by overeating, obesity, insulin resistance and diabetes, in particular It is given to animals with genetic background of C57BL / Ks. db / db mice are typically identified as obese at 3-4 weeks of age and plasma insulin elevations begin after 10-14 days. Glucose elevations were observed at 4-8 weeks with uncontrolled elevation of blood glucose and severe damage to insulin producing β cells in pancreatic islet cells, and died at about 10 months of age. This model has been used to characterize drug candidates' ability to influence the rate or onset of parameters related to the onset and duration of diabetes, such as hyperglycemia and weight gain.

Treatment of diabetes with PPAR-γ agonists involves cardiovascular hypertrophy or increased heart weight. Treatment with rosiglitazone maleate, a PPAR-γ agonist, indicates that the patient may experience fluid accumulation and volume related symptoms such as edema and hemorrhagic heart failure. Cardiac hypertrophy associated with PPAR-γ agonists is usually treated by discontinuing treatment.

The physiological effect of cortisol is antagonism of insulin. Higher levels of cortisol in the liver can reduce insulin sensitivity in the organs, leading to increased glucose gluconeogenesis and plasma levels (MF Dallman et al. Front Neuroendocrinol., 14: 303-347 , 1993). This effect exacerbates impaired glucose tolerance or diabetes. Cushing's syndrome is a disease caused by excessive plasma concentrations of cortisol. Insulin antagonism may cause diabetes in sensitive subjects (EJ Ross et al., Lancet, 2: 646-649,1982). ).

Cortisol can be converted to cortizone in the body by the 11β-dehydrogenase activity of the 11β-hydroxysteroid dehydrogenase enzyme. The reverse reaction of converting inactive cortisone to active cortisol is carried out in certain organs by the 11β-reductase activity of the enzyme. This activity is also known as corticosteroid 11β-reductase activity. There are at least two different isozymes in the 11β-hydroxysteroid dehydrogenase. Expression of type 1 11β-HSD in the cell line range generates bidirectional enzymes or mainly 11β-reductase, which can generate 11β-hydroxysteroids from an inactive 11-ketosteroid parent. have.

Mitochondrial phosphoenolpyruvate carboxykinase (also PEPCK-mitochondria, PEPCK-M, PCK2 and mtPEPCK) is expressed in various human tissues, mainly liver, kidney, pancreas, small intestine and fibroblasts (Modaressi et al., Biochem. J., 333: 359-366, 1998). Although not clear, PEPCK-mitochondrial deficiency is associated with growth deficiency, hypoglycemia and liver failure. Unlike the cytoplasmic form (PEPCK-C), the mitochondrial form (PEPCK-mitochondria) is constantly expressed and not regulated by hormonal stimulation (Hanson and Patel, Adv. Enzymol. Relat. Areas Mol. Biol., 69: 203 -281, 1994). The two forms are on separate chromosomes, one on chromosome 14q11 and PEPCK-C on chromosome 20q11 (Stoffel et al., Hum. Mol. Genet., 2: 1-4 , 1993).

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system, an autoimmune disease (Bar-Or, A., J. Neuroimmunol . 100: 252-259, 1999). Although natural progression of the disease has recently been found to be improved by treatment with immunomodulatory-immunosuppressive compounds such as interferon (IFN) -β, copolymers, cyclophosphamide and mitoxanthrone (Hafler, DA and Weiner, HL , Immunological Reviews 144: 75, 1995; Goodkin, DE, Lancet 352: 1486, 1998), none of these drugs can block the progression of the disease, and some of these drugs have serious side effects that limit their long-term use. Moreover, a substantial number of patients with relapsed and secondary advanced MS are less likely to respond to IFN-β. Therefore, there is a need for new compounds that improve the progression of MS alone or as a combination treatment, for example by slowing its progression.

There is a need for more cost effective pharmaceutical agents or more effective methods of treatment for treating the conditions described above. The present invention provides therapeutics and methods for treating one or more of these symptoms. Therefore, the therapeutic agents and methods of treatment are useful for reducing one or more symptoms associated with the symptoms described herein. In addition, the use of the therapeutic agents and methods of the present invention may be combined with one or more of the conventional therapies for such diseases.

Summary of Embodiments of the Invention

The present invention provides for the treatment, prevention, slowing down, or alleviation of undesirable inflammatory symptoms, autoimmune diseases or metabolic diseases or symptoms thereof in a mammal in need (optionally the mammal is a human or non-human primate). To provide a composition or compound, wherein the composition contains a compound of formula

In the above formulae, ^one of R ¹ is -H or optionally substituted alkyl, the other R ¹ is -H, -OH, ester or ether, or two R ¹ together are = O or = NOH or Oxime, ester or ether, such as = NOCH ₃ ; One of R ² is —H or optionally substituted alkyl, the other R ² is —H, —OH, an ester or an ether, or two R ² together = O, or = NOH or = NOCH _3, etc. Oxime of; One of R ³ is —H or optionally substituted alkyl, the other R ³ is —H, —OH, an ester or an ether or optionally substituted alkyl, or two R ³ together = O, or = Oximes such as NOH or = NOCH ₃ ; One of R ⁴ is —H or optionally substituted alkyl, the other R ⁴ is —OH, an ester or an ether, or two R ⁴ together represent an oxime such as = O, or = NOH or = NOCH ₃ Represent; R ⁵ is optionally substituted alkyl and is optionally selected from —CH ₃ , —C ₂ H _5, and —CH ₂ OH; R ⁶ is —H or optionally substituted alkyl, optionally selected from —CH ₃ , —C ₂ H _5, and —CH ₂ OH; One of R ⁷ is —H or optionally substituted alkyl and the other R ⁷ is —H, —OH, an ester or an ether, or when there is no double bond at position 4, the two R ⁷ are together Oxime, such as = O or = NOH or = NOCH ₃ ; R ⁹ is —O— or —C (R ¹² ) (R ¹² ) — wherein one of R ¹² is —H, —F, —Br or optionally substituted alkyl, and the other R ¹² is — H, —OH, an ester or an ether or optionally substituted alkyl, or two R ¹² together represent an oxime, such as ═O, or ═NOH or ═NOCH ₃ ; R ¹⁰ is -H or halogen such as -F or -Cl; One of R ¹¹ is —H or optionally substituted alkyl, the other R ¹¹ is —H, —OH, an ester or an ether or optionally substituted alkyl, or two R ¹¹ together are = O, or = NOH Or oxime, such as = NOCH ₃ . Embodiments of such compounds include (i) one or two or three of R ² , R ⁷ , R ¹¹ and R ¹² are independently —OH, C _2-8 ester, C _1-8 ether or _═O (ii) one or two of R ¹ , R ⁷ , R ¹¹ and R ¹² is —OH, C _2-8 ester or C _1-8 ether, and (iii) R ¹ , R ² , R ⁷ , One or two of R ¹¹ and R ¹² are ═O or = NOH, and one or two or three of the others are independently a compound that is —OH, a C _2-8 ester or a C _1-8 ether. do. When present, the hydrogen atom at the 5th position may be present in α or β steric configuration. One of the R ⁷ moieties is absent when the double bond is present at position 4.

The invention also identifies or characterizes the biological activity of a compound having the potential to treat or alleviate metabolic diseases in a mammal comprising selecting a compound having the characteristics of (i)-(iv) Also provided are methods, wherein the compounds having the characteristics of (i) to (v) are as follows: (i) in human or mammalian cells in vitro as compared to a suitable negative control human or mammalian cell in vitro; Compounds that do not activate more than about 30% of one, two or three of PPAR-α, PPAR-γ and PPAR-δ; (ii) compounds that inhibit or reduce the transcriptional activity or level of NF-κB in human or mammalian cells by about 20 to 80% in vitro as compared to a suitable negative control human or mammalian cell; (iii) reduced hyperglycemia, slowed the progression or delayed onset of hyperglycemia, increased insulin sensitivity, decreased glucose intolerance, decreased pancreatic β-islet cell number or their insulin as compared to the appropriate negative or normal control group. Slows the rate or progression of the ability to secrete, increases the pancreatic β-islet cell number or their insulin secretion ability, slows the rate of weight gain in db / db mice or feeding induced or obese subjects , Lowers elevated levels of triglycerides, lowers total blood or plasma cholesterol levels, lowers normal or elevated levels of blood or plasma LDL, VLDL, apoB-100 or apoB-48, or lowers blood or plasma Increases normal or low levels of HDL or apoA1, or elevated levels of fibrinogen in the blood or plasma Chu compound; And (iv) optionally a glucocorticoid receptor, a mineralocorticoid receptor, a progesterone receptor, an androgen receptor, an estrogen receptor-α, an estrogen receptor-β or a human in a mammalian cell, as compared to a suitable negative control human or mammalian cell in vitro. A compound that does not activate greater than about 30% of the activity of one or more of the biologically active variants of the biological molecule. The methods can identify or characterize compounds having the potential to treat or alleviate metabolic diseases in humans or other mammals.

Embodiments also include compositions comprising a compound of formula 1 for treating or preventing autoimmune symptoms, undesirable inflammatory symptoms or metabolic diseases or signs of such symptoms.

Other embodiments are described in detail in the following detailed description of the invention, including the embodiments described herein.

Justice

As used herein, unless otherwise indicated herein, these terms are used with the meanings defined herein. The description of the embodiments and examples described herein is for the purpose of describing the invention and is not intended to limit the invention in any way. Unless otherwise defined or described as such, for example, in the definition or throughout this specification, the singular forms "a," "an" and "the" are either singular or plural meanings unless otherwise stated or described as being mutually exclusive or optional. Included, the term "or" means "and / or".

"Formulation" or similar term means a composition that can be administered to a subject, such as a human or an animal. Formulations are suitable for use in humans or vertebrates and have general characteristics for their formulations, such as oral formulations for use in humans, for example, will usually be sterile solutions or suspensions.

The term “excipient”, “carrier”, “pharmaceutically acceptable carrier” or similar term is acceptable when used in combination with other components of the compositions or formulations of the present invention and includes the patient, animal, tissue or It means one or more components that are not harmful to the cell.

The term "subject" means a human or animal. Usually animals refer to mammals or non-human primates, rodents, rabbits, livestock or hunting animals. Primates include chimpanzees, cynomologus monkeys, spider monkeys and macaques, such as Rhesus or Pan monkeys. Etc. Rodents and rabbits include mice, rats, woodchucks, ferrets, rabbits and hamsters.

As used herein, "alkyl" refers to a linked normal, secondary, tertiary or cyclic carbon atom, ie can be linear, branched, cyclic or any combination of the above. As used herein, an alkyl moiety may be saturated or unsaturated, ie, the alkyl moiety may comprise one, two or more independently selected double bonds or triple bonds. Unsaturated alkyl moieties include moieties as those described for the alkenyl and alkynyl moieties described below. The number of carbon atoms in an alkyl group or alkyl moiety is 1 to about 50, for example about 1 to 30, or about 1 to 20, and unless otherwise specified, for example C _1-8 alkyl is 1 Or an alkyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms. When an alkyl group is mentioned, the species includes methyl, ethyl, 1-propyl ( n -propyl), 2-propyl ( i -propyl, -CH (CH ₃ ) ₂ ), 1-butyl ( n -butyl), 2- Methyl-1-propyl ( i -butyl, -CH ₂ CH (CH ₃ ) ₂ ), 2-butyl ( s -butyl, -CH (CH ₃ ) CH ₂ CH ₃ ), 2-methyl-2-propyl ( t -Butyl, -C (CH ₃ ) ₃ ), 1-pentyl ( n -pentyl), 2-pentyl (-CH (CH ₃ ) CH ₂ CH ₂ CH ₃ ), 3-pentyl (-CH (CH ₂ CH ₃₎ ) ₂ ), 2-methyl-2-butyl (-C (CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH (CH ₃ ) CH (CH ₃ ) ₂ ), 3-methyl -1-butyl (-CH ₂ CH ₂ CH (CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH (CH ₃ ) CH ₂ CH ₃ ), 1-hexyl, 2-hexyl (-CH (CH ₃ ) CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH (CH ₂ CH ₃ ) (CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C (CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH (CH ₃ ) CH (CH ₃ ) CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH (CH ₃ ) CH ₂ CH (CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C (CH ₃ ) (CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH (CH ₂ CH ₃ ) CH (CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C (CH ₃ ) ₂ CH (CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH (CH ₃ ) C (CH ₃ ) ₃ ), cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,-(CH ₂ ) _n- (CHCH ₃ ) _m- (CH ₂ ) _o -CH ₃ and-(CH ₂ ) _n- (CHC ₂ H ₅ ) _m- (CH ₂ ) _o -CH ₃ , wherein n, m and o are independently 0, 1, 2, 3, 4, 5, 6, 7 or 8).

As used herein, “alkenyl” refers to one or more double bonds (eg, —CH═CH—), such as one, two, three, four, five, six or more, By moiety comprising a linked normal, secondary, tertiary or cyclic carbon atom usually comprising one or two, ie linear, branched, cyclic or a combination of the foregoing. The number of carbon atoms in the alkenyl group or alkenyl moiety is 2 to about 50, for example about 2 to 30, or about 2 to 20, unless otherwise specified, for example C _{2- 8} alkenyl or C2-8 alkenyl means an alkenyl moiety containing 2, 3, 4, 5, 6, 7, or 8 carbon atoms. When an alkenyl group is mentioned, the kind includes vinyl, allyl,-(CH ₂ ) _n- (CH = CH)-(CH ₂ ) _m -CH ₃ ,-(CH ₂ ) _n- (CCH ₃ = CH)- (CH ₂ ) _m -CH ₃ ,-(CH ₂ ) _n- (CH = CCH ₃ )-(CH ₂ ) _m -CH ₃ and-(CH ₂ ) _n- (CH = CH) _0-1- (CH ₂ ) _m -CH ₂ CH = CH ₂ , where n and m are independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

As used herein, “alkynyl” refers to one or more triple bonds (—C≡C—) such as one, two, three, four, five, six or more, usually one or two. Dogs, optionally including one, two, three, four, five, six or more double bonds, the remaining bond being a single bond, linked normal, secondary, tertiary or inter By moiety comprising a click carbon atom, ie, linear, branched, cyclic, or any combination of the foregoing. The number of carbon atoms in the alkenyl group or alkenyl moiety is 2 to about 50, unless otherwise specified, for example about 2 to 30 or about 2 to 20, for example C _2-8 alky Nyl or C2-8 alkynyl means an alkynyl moiety containing two, three, four, five, six, seven or eight carbon atoms. When an alkynyl group is mentioned, the alkynyl group and species include -CCH, -CCCH ₃ , -CCCH ₂ CH ₃ , -CCC ₃ H ₇ , -CCCH ₂ C ₃ H ₇ ,-(CH ₂ ) _n- (C _{≡C) - (CH 2) m} -CH 3 and _{- (CH 2) n - (} C≡C) 0-1 - ( will contain CH _{2) m} -CH ₂ C≡CH, where n and m are independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

Terms such as "substituted alkyl", "substituted alkenyl", "substituted alkynyl" include substituents linked to carbon atoms while replacing hydrogen atoms, or substituents that interrupt a carbon atom chain, or Having an alkyl, alkenyl, alkynyl or other functional group or moiety as described herein. Substituents are -F, -Cl, -Br, -I, -OH, -OR ^PR , -SH, -SCH ₃ , -O-, -S-, -NH-, -C (O)-, -C ( O) OR ^PR , -CHO, -CH ₂ SH, -C = N-, -C (O) OR ^PR , -C (O) CH ₃ , wherein R ^PR is independently hydrogen, a protecting group, or two R ^PR represents hydrogen or together a protecting group), one, two, three, four, five, six or more groups independently selected.

"Halogen" means fluorine, chlorine, bromine or iodine.

“Ester” means a moiety that includes a —C (O) —O— structure. Usually, the esters used in the present invention contain about 1 to 50 carbon atoms (eg, about 2 to 20 carbon atoms) and 0 to about 10 independently selected heteroatoms (eg, O, S, N , P, Si), wherein the organic moiety is bonded to R ¹ or R ² of the steroid nucleus of Formula 1, for example, through a -C (O) -O- structure For example organic moiety-C (O) -O-steroids or organic moiety-OC (O) -steroids. The organic moiety is usually the aforementioned organic functional group, for example C _1-20 alkyl moiety, C _2-20 alkenyl moiety, C _2-20 alkynyl moiety, aryl moiety, C _2-9 heterocycle Or substituted derivatives thereof, eg, one or more derivatives comprising one, two, three, four or more substituents, wherein each substituent is independently selected. The esters are succinic acid esters, dicarboxylic acid esters and amino acid esters, such as -OC (O)-(CH ₂ ) _n -C (O) -OR ^PR , -OC (O)-(CH ₂ ) _n -NHR ^PR and —NH— (CH ₂ ) _n —C (O) —OR ^PR , where n is 1, 2, 3, 4, 5, 6, 7 or 8 and R ^PR is —H or a protecting group such as C 1-4 alkyl It is included). The ester also includes structures such as -OC (O) -O- (CH ₂ ) _n -H and -OC (O) -NH- (CH ₂ ) _n -H.

Exemplary substituents on hydrogen or carbon atoms in these organic functional groups are the same as described for the alkyl moieties described above, and 1, 2, 3, 4, 5, 6 or more, usually 1 , 2 or 3 -O-, -S-, -NR ^PR- (including -NH-), -C (O)-, -CHO, -CHS, -C = NH, -C (S), = O, = S, -N (R ^PR ) ₂ (with -NH ₂ ), -C (O) OR ^PR (with -C (O) OH), -OC (O) R ^PR (-OC (O) -H), -OR ^PR (with -OH), -SR ^PR (with -SH), -NO ₂ , -CN, -SCN, -C ₆ H ₅ , -CH ₂ C ₆ H ₅ , -NHC ( O)-, -C (O) NH-, -OC (O)-, -C (O) O-, -O-A8, -S-A8, -C (O) -A8, -OC (O) -A8, -C (O) O-A8, = N-, -N =, = N-OH, -OPO ₃ (R ^PR ) ₂ , -OSO ₃ H ₂ or a halogen moiety or atom (where each R ^PR Is -H, an independently selected protecting group or two R ^PR together comprise a protecting group, A8 is C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-4 alkyl-aryl (e. g., benzyl), aryl (e.g., phenyl) or C _0-4 alkyl, -C _2-9 heterocyclic It includes a). Substituents are independently selected. Organic moieties include those in the compounds defined for R ₄ . Organic moieties do not include apparently unstable moieties, for example moieties such as -OO-, provided that such unstable moieties are described herein, including the use of synthetic compounds of Formula 1 and other compounds. For the above uses, this is not the case for temporary varieties that can be used to make compounds with sufficient chemical stability. Substituents listed above are usually substituents that can be used to replace one or more carbon atoms, such as -O- or -C (O)-, or substituents that can be used to replace one or more hydrogen atoms, such as halogen,- NH ₂ or -OH. Exemplary esters include one or more independently selected acetate, enanthate, propionate, isopropionate, cyclopropionate, isobutyrate, n-butyrate, valerate, caproate, isocaproate, hexanoate, hep Tanoate, octanoate, nonanoate, decanoate, undecanoate, phenylacetate or benzoate, usually hydroxyl esters. Esters also include amino acids, carbonates, and carbamates, including -OC (O) -CH ₂ -NHR ^PR , -OC (O) -CH ₂ CH ₂ -NHR ^PR , -OC (O) -CH (CH ₃ ) -NHR ^PR , -OC (O) -CH ₂ CH (CH ₃ ) -NHR ^PR , -OC (O) -CH (NHR ^PR ) -CH (OR ^PR ) -CH ₃ , -OC (O) -O -(CH ₂ ) _m -H and -OC (O) -NH- (CH ₂ ) _m -H wherein R ^PR is -H or C _1-4 alkyl (-CH ₃ , -C ₂ H ₅ ,- C ₃ H _7, etc.) or a protecting group such as -C (O) -CH ₃ or -CH ₂ CH ₂ -O-CH ₃ , and m is 0, 1, 2, 3, 4, 5 or 6). Ester includes -OC (O)-(CF ₂ ) _n -CF ₃ , -OC (O)-(CH ₂ ) _n -CH ₃ , -OC (O) -CH (CH ₃ )-(CH ₂ ) _n- CH ₃ and —OC (O) —C (CH ₃ ) ₂ — (CH ₂ ) _n —CH _{3 are} also included, wherein n is 0, 1, 2, 3, 4, 5 or 6.

"Ether" means the same organic moiety as the description for the ester, except that it contains one, two, three, four or more, usually one or two -O- moieties. do. In some embodiments, the -O-functional group is linked to the steroid nucleus at various groups, such as R ¹ , R ² , R ³ , R ⁴ or R ¹¹ , for example an organic moiety-O-steroid. do. The organic moiety is the same as described for the ester. Ethers include -O- (CH ₂ ) _n -CH ₃ , -O-CH ₂ (CH ₂ ) _n -O-CH ₃ , -O-CH ₂ (CH ₂ ) _n -S-CH ₃ and -O-CH (CH ₃ )-(CH ₂ ) _n -CH ₃ , where n is 0, 1, 2, 3, 4, 5 or 6, is also included.

Compound of Formula 1

In some embodiments, the compound of formula 1 comprises three, four or five hydroxy groups, optionally one, two or more are esterified with the same or different ester groups. In some such embodiments, the hydroxyl group or ester is in the 3-, 4-, 16- and 17-positions, wherein the hydroxyl group or ester in the 3-, 4-, 16-position is β, β, respectively. , α, β, β, β, α, β, α, α, β, β, β, α, α, β, α, β, α, α, α or α, α, β in a three-dimensional arrangement. The hydroxyl or ester in the 17-position is usually present in the β-stereo array, but may also be present in the α-stereo array. R ¹⁰ of this compound is usually halogen such as -H or -F, R ⁵ is optionally -CH ₃ or -C ₂ H ₅ , and R ⁶ is optionally -H or -CH ₃ . For some of these compounds additional hydroxyl groups or esters may be present at the 7- or 11-position of the β-stereo array or the α-stereo array.

For compounds of formula 1, substituted alkyl moieties of C _1-8 include —CH ₂ F, —CF ₃ , —CH ₂ OH, and —C ₂ F ₅ . C _2-8 optionally substituted alkynyl moieties include —CCH, —CCCH ₃ , —CCCH ₂ OH, —CCCH ₂ Cl and —CCCH ₂ Br.

Exemplary compounds of Formula 1 include androast-5-ene-3β, 4β, 7β, 16α, 17β-pentol and epimers of the compound, wherein the epimer has one or two hydroxyl groups Since it is mercurized, it is for example androst-5-ene-3α, 4β, 7β, 16α, 17β-pentol, androast-5-ene-3β, 4α, 7α, 16α, 17β-pentol and androst-5 -N-3β, 4β, 7α, 16β, 17β-pentol. In the other compounds of formula 1 are five independently selected -OH, ester or ether moieties such as androst-5-ene-3β, 4β, 11β, 16α, 17β-pentol and one Or epimers of this compound wherein two hydroxyl groups are epimerized, such as androst-5-ene-3α, 4β, 11β, 16α, 17β-pentol, androast-5-ene-3β, 4β, 11β, 16α, 17α-pentol, androast-5-ene-3β, 4β, 11α, 16α, 17β-pentol and androast-5-ene-3β, 4α, 11β, 16β, 17β-pentol. For such compounds, one, two or more of the five hydroxyl groups can be derivatized, for example methoxy, ethoxy, acetoxy, propionoxy, -OC (O)- Or ethers such as (CH ₂ ) ₃ —H, —OC (O) — (CH ₂ ) ₄ —H, or —OC (O) — (CH ₂ ) ₅ —H derivatives. Other esters include -OC (O) -CH ₂ -C (O) OH, -OC (O)-(CH ₂ ) ₂ -C (O) OH, -OC (O)-(CH ₂ ) ₃ -C ( O) OH, -OC (O)-(CH ₂ ) ₄ -C (O) OH, -OC (O)-(CH ₂ ) ₅ -C (O) OH and -OC (O)-(CH ₂ ) _6- C (O) OH.

In some such embodiments, four independently selected hydroxyl, ester or ethers are at four of the 2-, 3-, 4-, 7-, 11-, 16- and 17-positions of the compound of Formula 1 exist. Such substituents are for example 2-, 3-, 16-, 17-position, 2-, 3-, 7-, 17-position, 2-, 3-, 11-, 17-position, 3-, 4- , 7-, 17-position, 3-, 4-, 11-, 17-position, 3-, 7-, 16-, 17-position, 3-, 4-, 16-, 17-position or 3-, May be connected to the 11-, 16-, 17-positions. Hydroxyls, esters or ethers are 2- and / or 3-β, β, β, β-17, 2- and / or 3-β, β, respectively, when a double bond is not present in the 4-position, β, α-17, 2- and / or 3-β, β, α, β-17, 2- and / or 3-β, α, β, β-17, 2- and / or 3-α, β , β, β-17, 2- and / or 3-β, β, α, α-17, 2- and / or 3-β, α, β, α-17, or 2- and / or 3-α , β, β, α-17 may exist in three-dimensional configuration. The term "2- and / or 3-β, β, β, β-17" means that the 2-position is optionally substituted and the 3- and 17-positions are substituted with hydroxyl, ester, or ether. Hydroxyls, esters or ethers are 2- and / or 3-β, α, α, β-17, 2- and / or 3-α, β, respectively, when a double bond is not present in the 4-position, α, β-17, 2- and / or 3-α, α, β, β-17, 2- and / or 3-β, α, α, α-17, 2- and / or 3-α, β , α, α-17, 2- and / or 3-α, α, β, α-17, 2- and / or 3-α, α, α, β-17, 2- and / or 3-β, may exist in α, α, α-17 conformations. R ¹⁰ of this compound is usually -H or -F, R ⁵ is optionally -CH ₃ or -C ₂ H ₅ , and R ⁶ is optionally -H, -CH ₃ , -CH ₂ OH, -CCH or CCCH ₃ . For this compound, one, two or more of the 2-, 3-, 4-, 7-, 11-, 16- and 17-positions is -H or -CH ₃ , -C ₂ H ₅ , Substituted with optionally substituted alkyl, such as —CH ₂ ═CH ₂ , —CCH, —CF ₃ or —C ₂ F ₅ .

In some embodiments of compounds of Formula 1, there may be five independently selected hydroxyl, ester and / or ether moieties. For these compounds, the hydroxyl, ester and / or ether moieties are usually present in the 3- and 17-positions and three other positions. These substituents may be present at 2-, 3-, 7- 11-, 17-position, 2-, 3-, 7-16-, 17-position or 2-, 3-, 11-16-, 17-position have. Independently selected hydroxyl, ester and / or ether moieties are 3-, 4-, 7-11-, 17-position, 3-, 4-, 11-16-, 17-position, 3-, 4-, It may be present at the 7-16-, 17-position or 3-, 7-, 11-, 16-, 17-position. In this compound, each moiety may be present in an α-stereo array or in a β-stereo array when the double bond is not present in the 4-position. Therefore, the substituents in the compounds are 2- and / or 3-β, β, β, β, β-17, 2- and / or 3-β, β, β, β, α-17, 2- and / or 3-β, β, β, α, β-17, 2- and / or 3-β, β, α, β, β-17, 2- and / or 3-β, α, β, β, β- 17, 2- and / or 3-α, β, β, β, β-17, 2- and / or 3-β, β, β, α, α-17, 2- and / or 3-β, β , α, β, α-17, 2- and / or 3-β, α, β, β, α-17, 2- and / or 3-α, β, β, β, α-17, 2- and And / or 3-β, β, α, α, β-17, 2- and / or 3-β, α, β, α, β-17, 2- and / or 3-α, β, β, α, β-17, 2- and / or 3-β, α, α, β, β-17, 2- and / or 3-α, β, α, β, β-17, 2- and / or 3-α , α, β, β, β-17, or 2- and / or 3-β, β, α, α, α-17 may be present in stereo configuration, respectively. Substituents of these compounds may also be 2- and / or 3-β, α, β, α, α-17, 2- and / or 3-β, α, α, β, α-17, 2- and / or 3-β, α, α, α, β-17, 2- and / or 3-α, β, β, α, α-17, 2- and / or 3-α, β, α, β, α- 17, 2- and / or 3-α, β, α, α, β-17, 2- and / or 3-α, α, β, β, α-17, 2- and / or 3-α, α , β, α, β-17, 2- and / or 3-α, α, α, β, β-17, 2- and / or 3-β, α, α, α, α-17, 2- and And / or 3-α, β, α, α, α-17, 2- and / or 3-α, α, β, α, α-17, 2- and / or 3-α, α, α, β, α-17, 2- and / or 3-α, α, α, α, β-17, or 2- and / or 3-α, α, α, α, α-17 stereoscopic configurations.

In the compounds of Formula 1 described herein, the 16-position may be unsubstituted, that is, the first or all R ³ is —H, and the second R ⁴ is free of double bonds at the 17-position In the case of α-stereo array or β-stereo array, it may be present at the 17-position. The first 2 R ⁴ moiety is _{-CH 3, -C 2 H 5,} -CF 3, -C 2 F 5, -C = CH 2, -CCH, -CCCH 3 , or a C _1- -CCCH ₂ OH, etc. ₈ optionally substituted alkyls are included.

In this compound, the 2-position may be unsubstituted, that is, R ⁹ is -CH _2- . In some embodiments, R ⁹ is substituted, for example -O-, -CH (OH)-, -CH (ester)-or -CH (ether)-, wherein the hydroxyl, ester or ether moiety is α- or β-stereo array). The R ⁹ moiety is -CH (α-OH)-, -CH (β-OH)-, -C (β-CH ₃ ) (α-OH)-, -C (α-CH ₃ ) (β-OH )-, -CH (α-OCH ₃ )-, -CH (β-OCH ₃ )-, -CH (α-OC (O) CH ₃ )-and -CH (β-OC (O) CH ₃ )- to be. Other R ⁹ moieties include -CH (α-OC (O) CH ₂ CH ₃ )-, -CH (β-OC (O) CH ₂ CH ₃ )-, -CH (α-OCH ₂ CH ₃ )-, -CH (β-OCH ₂ CH ₃ )-, -C (β-CH ₃ ) (α-OC (O) CH ₃ )-and -C (α-CH ₃ ) (β-OC (O) CH ₃ ) -to be.

Biodynamic compounds

Methods for identifying or characterizing compounds, including biomechanical compounds, can be performed as described above. The method further includes conducting a protocol to determine whether the test compound modulates the activity or level of the mediator of an acute biological response in an in vitro assay by about 20% or about 25% to about 70% or about 75%. And optionally wherein said test compound activates more than about 10%, about 20%, or about 30% of the activity of the glucocorticoid receptor when compared to a suitable reference active agent or antagonist of a glucocorticoid receptor such as dexamethasone or cortisol, for example. And performing a protocol to determine whether or not to antagonize. In this embodiment, the subject may be exposed to acute stimulation or biological injury, i.e., to a sufficient amount of ionizing radiation or pre-inflammatory signal, compound or composition, wherein optionally the pre-inflammatory signal, compound or composition Is bacterial LPS or TNFα, and / or optionally the mediator of an acute biological response is NF-κB or IκB.

Acute stimulation or bioinjury is a test for mediators of acute biological responses at the time of (i) and (ii) following administration of sufficient bacterial LPS to a sufficient number of drug treated mice and a sufficient number of vehicle control mice. It may be to determine the effect of the compound, wherein the time point of (i) is the point at which the acute response is at or near the maximum, optionally about 1.5 hours after administration of bacterial LPS by intraperitoneal injection, for example about 70 To 110 minutes or 75 to 105 minutes, wherein (ii) is one or two other time points before and / or after administration of sufficient bacterial LPS, optionally one time point before administration of sufficient bacterial LPS And at one time point after the acute response is at or near the maximum, optionally about 2.0 or 2.5 hours after administration of bacterial LPS by intraperitoneal injection, Optionally a mediator of acute biological response is NF-κB, or IκB.

The administration of sufficient bacterial LPS may optionally be carried out substantially in accordance with the methods described herein, or with modifications thereof, optionally with the ability of the compound to partially modulate the level or activity of the mediator of an acute biological response. Substantially according to the method described in or a suitable modification thereof.

Other irritants or biological injuries of interest include ischemia and reperfusion of one or more ischemic tissues, relatively low, moderate or high severity thermal or chemical burns, or exposure to other toxins or toxins. The effects of biological injury can be measured in various tissues or organs, such as the spleen, blood, spinal cord, lungs, brain, liver, small intestine, colon or heart.

Variables that can be measured usually include the time or period of time that the subject responds maximally to a given biological injury. In general, acute biological injury will cause a maximum biological response within the first 30 minutes to 48 hours after subject exposure to the injury. The maximum response by a given biomolecule to an acute biological injury will generally appear between about 15 minutes and about 2 hours at the latest, but some reactions may begin after a period of time and then form for a longer period of time. The maximum duration of the biological response is a convenient period that allows the drug mechanisms of action or efficacy to be measured.

The ability of 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol to be transient, but very potent, effective and later disappear and return to normal function is a biodynamic response in the present invention. (See Example 9). The biomechanical reactions induced by biomechanical agents, such as 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, are contrasted with the 'biostatic reaction'. , Dexamethasone and the like are reactions that cause glucocorticoid receptors or their effector biomolecules such as NF-κB, which are indirectly inhibited through activation of glucocorticoid receptors. A biostatic response is essentially an 'all on all the time' response to the biological efficacy of a 'biostatic agent', such as dexamethasone, with relatively little change in target cells or tissue at a given concentration. .

Therefore, the pharmacokinetic effects of the biostatic preparations vary mainly with the pharmacokinetic properties or their concentration. Conversely, the characteristics of biomechanical agents, such as 7α-ethynylandrost-5-ene-3β, 7β, 17β-triol, are characterized by the nature and intensity underlying the concentration and biological stimulation in the target cell or tissue. It is a pharmacokinetic effect affected by the combination. Thus, for example, by exposure to potentially lethal amounts of ionizing radiation such as gamma rays or X-rays, or by activating or inhibiting mediators of bacterial LPS, TNFα or other NF-κB, IκB, IL-6, C reactive proteins Biological irritation is induced by exposure to a formulation which may be present. In this regard, biomechanical drugs may have lower statistical interrelationships or less significant relationships between pharmacokinetic and pharmacokinetic effects as compared to those generally observed with biostatic drugs.

One aspect of biomechanical drugs is their potential ability to reduce systemic toxicity associated with biostatic drugs that may act at least in part through modulating the same or similar target biomolecules. Biomechanical drugs, such as dexamethasone, can be used clinically to treat a wide range of inflammatory symptoms, but all bioactivity at all times can cause toxicity. In the case of inhibiting NF-κB, constant and relatively complete inhibition of its activity, for example about 75%, 80%, 85%, 90%, 95% or essentially 100% in most or all tissues Inhibiting long periods of time, for example from 1, 2, 4 hours to about 1, 2, 3 days or more, is undesirable because some basic NF-κB activity is required for normal biological function in most tissues. May cause side effects. Known toxicity associated with the use of glucocorticoids, such as dexamethasone, appears to result at least in part from the relatively complete shut down of affected biological molecules such as NF-κB. In contrast, biomechanical drugs can elicit more transient responses that can alleviate or reduce undesirable toxic side effects.

Another aspect of biomechanical drugs is their ability to potentially exert therapeutic effects in a tissue specific manner. Therefore, the animal's response to challenge, such as reperfusion to affected tissues after transient ischemia or exposure to biological injuries such as potentially lethal amounts of bacterial LPS, may indicate the extent of activation of NF-κB in various tissues. This can be proved by diversifying. Biomechanical drugs act on tissues in which the animal's response is relatively large, such as the mouse spleen or myocardium, while the target organism, at least in part, mediates the response to biological injury, such as in other tissues, such as the brain. In other tissues that are relatively lower than the molecule, they remain functional without relatively affecting the function of NF-κB.

Biomechanical drugs may act in part by their ability to partially inhibit their target biomolecules. As also described in Example 7, 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol and some other compounds described partially inhibited the activity of NF-κB in cells in vitro. However, no complete inhibition was observed at any concentration. This is in contrast to the activity of the biostatic drug dexamethasone, which completely inhibits the activity of NF-κB at sufficiently high concentrations. Partial inhibition of NF-κB in this in vitro seems to be partially reflected by its activity described in this example. Therefore, in at least some cases, a feature of biomechanical drugs is that they have the ability to partially inhibit or activate target biomolecules in systems such as in vitro assays as described in Example 7. Inhibition of NF-κB appears to be indirect because 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol and the other compounds described herein are NF-κB in the cytoplasm or nucleus. I don't think it's combined directly with.

The protocol described in this example, or a suitable modification thereof, can be used as a biomechanical or biostatic agent by modulating (detectably activating or detectably antagonizing or detectably inhibiting) molecules such as NF-κB in vivo. It can be used to characterize other compounds for their ability to act. This compound may also be analyzed by in vitro assays, such as those described in Example 7, to further analyze the mechanism of action. Suitable variations of the in vivo protocol described in this example include varying dosages of test compounds and varying methods of administration, for example oral, buccal, sublingual or intradermal, subcutaneous, intravenous or About 0.1 mg when administered parenterally, such as intramuscular injection, or by intranasal or inhalation into the alveolar, alveolar, or alveolar or airway passages, e. / kg to about 350 mg / kg dosage range. Suitable test dosages are usually about 1 to 150 mg / kg. In vivo measurable biomolecules will be kinases such as IκB, src kinases, map kinases, or other signal mediators described herein. Such characterization methods can be performed in one or more subject ranges, such as, for example, rodents such as rats, non-human primates such as dogs, rhesus or rhesus monkeys. An animal group consisting of about 3 to 12 animals per group, e.g. 4 to 8 animals per group, can be used in a suitable vehicle or placebo control group, a test compound that is a potential biomechanical drug, 17α-ethynylandrost-5- It can be used in conjunction with a positive biomechanical drug control such as N-3β, 7β, 17β-triol, or a positive biomechanical drug control such as dexamethasone, wherein one or more controls can control the response of the test compound in different cell populations or tissues. For example, vehicle control, positive or negative biomechanical drug control or positive or negative biomechanical drug control.

When such an analysis is applied to humans, the range of experimental options will naturally be narrow compared to other animals. Human tissue or samples, such as blood, bone marrow, or lavage fluid, will be much easier to analyze than other tissue types such as the spleen or liver that must be obtained through invasive techniques. Therefore, it would be common to conduct animal studies prior to or performing human response measurements.

Inflammation treatment

An aspect of the activity of the compound of formula (1) is to reduce inflammation by affecting the mediators of inflammation such as NF-κB, IL-6 or TNFα. NF-κB molecules are usually important mediators of inflammation. Increased activity of NF-κB is associated with a range of inflammatory diseases and autoimmune symptoms.

The compound of formula 1 can be used to treat or alleviate symptoms associated with inflammation such as pain, fever or fatigue. Endometriosis; Heat; Rheumatoid fibritis; Glomerulonephritis; Rejection of a host's disease, organ or tissue graft for a graft, eg, kidney, lung, bone marrow or liver transplant rejection; Hemorrhagic shock; Rheumatoid fibrous histitis; Hyperalgesia; Inflammatory bowel disease; gastritis; Irritable bowel syndrome; Ulcerative colitis; Pepsin ulcers; Stress ulcers; Hemorrhagic ulcers; Hyperacidity; Dyspepsia; Stomach palsy; Gastroesophageal reflux disease; Inflammatory diseases of the joints, including osteoarthritis, psoriatic arthritis and rheumatoid arthritis; Inflammatory eye disease, such as may be associated with corneal transplant; Ischemia, including cerebral ischemia (eg, brain wounds that may occur as a result of trauma, epilepsy, bleeding or stroke, each of which can induce neurodegeneration); Kawasaki's disease; Poor learning; Lung disease (eg, ARDS); Multiple sclerosis; Muscle disease (eg, muscle protein metabolism, especially sepsis); Neurotoxins (eg, caused by HIV); osteoporosis; Pain, including cancer-related pain; Parkinson's disease; Alzheimer's disease; Periodontal disease; Early delivery; psoriasis; Reperfusion wounds; Septic shock; Side effects from radiation therapy; Transient lower jaw joint disease; Alcohol-induced liver wounds including alcoholic cirrhosis; Rheumatic fever; Sarcoidosis; Scleroderma; Chronic fatigue syndrome; Cardiovascular diseases and conditions, including hemorrhagic heart failure, coronary artery stenosis, myocardial infarction, myocardial dysfunction (eg, associated with sepsis), and coronary bypass grafts; Sleep disorders; Uveitis; Negative serum polyarthritis; Ankylosing spondylitis; Reiter's syndrome and reactive arthritis; Still's disease; Psoriatic arthritis; Enteropathic arthritis; Polymyositis; Dermatitis; Scleroderma; Systemic sclerosis; Vasculitis (eg, Kawasaki disease); Inflammation resulting from, for example, strains, sprains or cartilage damage; Wound recovery; Thin, fragile skin; Bleeding or ecchymosis; Erythema; And trauma. Trauma includes injury to tissues or organs associated with surgery such as wounds, chemical burns, thermal burns, radiation burns, and orthopedic surgery or abdominal surgery.

Inflammatory symptoms may include wounds due to reperfusion, restenosis after vascular surgery, myocardial infarction or inflammation associated with cerebral infarction. Unfavorable inflammatory symptoms or symptoms include pulmonary inflammatory symptoms such as cystic fibrosis, acute asthma, chronic asthma, steroid resistant asthma, acute bronchitis, chronic bronchitis, pulmonary pneumoconiosis, psoriasis, eczema, adult respiratory distress syndrome (ARDS) Or chronic obstructive pulmonary disease (COPD).

Autoimmune symptoms

Compounds of Formula 1 (compounds of Formula 1) and compositions described herein may be used to treat and prevent autoimmune diseases such as type 1 diabetes, Crohn's disease, arthritis, contact dermatitis, lupus and multiple sclerosis (MS) symptoms. It can be used to delay or slow down the process. MS symptoms include recurrent MS and secondary progressive MS. Lupus symptoms include systemic lupus erythematosus, lupus erythema related arthritis, lupus erythema related skin changes, lupus erythema related blood abnormalities, lupus erythema related kidney damage, lupus erythema related heart or lung disease, lupus erythema related neuropsychiatric changes, lupus erythema related Tissue inflammation, discoid lupus erythema, subcutaneous dermal lupus erythema and drug-induced lupus erythema. Arthritis and related symptoms include rheumatoid arthritis, osteoarthritis, fibromyalgia, primary osteoarthritis, secondary osteoarthritis, psoriatic arthritis, lupus erythema related arthritis, arthritis associated with acute or chronic inflammatory bowel disease or colitis, arthritis associated with ankylosing spondylitis, Tissue inflammation, joint pain, joint stiffness, joint movement damage, joint edema, joint inflammation, and synovial inflammation associated with arthritis.

A compound of Formula 1 or a compound containing Formula 1 and one or more excipients may treat symptoms such as ankylosing vertebralitis, psoriasis, eczema, colitis, Crohn's disease, acute or chronic inflammatory bowel disease, autoimmune kidney wounds and liver wounds. Can be used to prevent, delay the onset, or slow the progression. Compounds of formula (1) include asthma symptoms, such as steroid-independent asthma, severe asthma, atopic asthma, acute or chronic asthma, allergic rhinitis, chronic bronchitis, acute bronchitis, cystic fibrosis, emphysema, pulmonary fibrosis, pulmonary airway hyperreactivity, It can be used to treat lung and airway-related symptoms, such as chronic obstructive pulmonary disease, pulmonary edema and acute respiratory distress syndrome.

Experimental autoimmune encephalomyelitis (EAE) refers to the experimental state of animals with clinical, histological and immunological characteristics similar to human MS, such as MS, in which T cells and monocytes are infiltrated into the CNS. EAE can induce sensitive mice by immunizing with proteolipid lipoprotein (PLP) in a suitable adjuvant. EAE animal models are in vivo models of human MS used to study the pathological mechanisms of MS and to characterize new agents for treating MS.

Treatment of Metabolic Diseases

Compounds of Formula 1 include type 1 diabetes, type 2 diabetes, X syndrome, hypercholesterolemia, hyperglycemia, insulin resistance (eg, associated with obesity), glucose intolerance, hypertriglyceridemia, hyperlipoproteinemia, fat It can be used to treat or prevent metabolic diseases such as dystrophy symptoms, X syndrome, arteriosclerosis, atherosclerosis, and obesity, or to slow the progression. Syndrome X (including metabolic syndrome) is associated with hyperinsulinemia, obesity, triglycerides, uric acid, fibrinogen, small high density LDL particles and elevated levels of plasminogen activator inhibitor 1 (PAI-1), reduced levels of HDL-c. It is defined as having two or more abnormalities. Many patients who have insulin resistance but have not yet progressed to type 2 diabetes are at risk of developing metabolic syndrome, ie, X syndrome, insulin resistance syndrome, or plurimetabolic syndrome. Syndrome X is a condition in which the patient has two or more of hyperlipidemia, hyperinsulinemia, obesity, insulin resistance, insulin resistance causing type 2 diabetes, and diabetes complications thereof, namely diseases in which insulin resistance is part of the etiology. Onset.

Cardiovascular diseases associated with metabolic diseases that can be treated with a compound of Formula 1 are associated with independent risk factors. These risk factors include high blood pressure, elevated fibrinogen levels, high levels of triglycerides, elevated LDL cholesterol, elevated total cholesterol levels and low HDL levels. Treatment can lead to stimulating pancreatic β cells to secrete more insulin and / or slowing the rate of pancreatic β cell loss that can occur over time in patients with diabetes or obesity.

Treating metabolic diseases using a compound of Formula 1 can be combined with other therapies. Diabetes can be treated using one or more of a variety of therapeutic agents, including compounds of Formula 1 and insulin sensitizers, including: insulin sensitizers such as PPAR-γ agonists such as glitazones; Biguanides; Protein tyrosine phosphatase-1B inhibitors; Dipeptidyl peptidase IV inhibitors; insulin; Insulin mimetics; Sulfonylureas; Meglitinides; α-glucoside hydrolase inhibitors; And α-amylase inhibitors. Metformin, phenformin, acarbose and rosiglitazone have been used to treat some types of diabetes.

As noted above, compositions containing a compound of Formula 1 can be used to treat, prevent, or slow the progression of, insulin resistance or symptoms of insulin resistance. Insulin resistance refers to a decrease in the biological activity of insulin, such as over a wide range of concentrations, as insulin exhibits a lower biological effect than expected. Subjects with insulin resistance cannot have adequate metabolism of glucose and have a reduced ability to, if not all, respond properly to insulin treatment. Symptoms of insulin resistance include insufficient glucose activation in muscle, insufficient insulin activation of oxidation and storage, inadequate insulin inhibition of lipolysis in adipose tissue, and inadequate insulin inhibition of glucose production and secretion in cells. Insulin resistance can lead to polycystic syndrome, impaired glucose tolerance, diabetes during pregnancy, high blood pressure, placenta and atherosclerosis. Compound compositions of formula 1 can be used to lower triglyceride levels in patients who are insulin resistant.

As noted above, embodiments of the present invention have potential potential for treating metabolic diseases or symptoms of humans or other mammals, slowing progression, slowing the onset of the disease, or alleviating the disease. Also included are methods for identifying compounds having (or "test compounds"). Compounds identified by this method are generally described as inerts of PPARs in vitro with one or more of the above-described activities, as incomplete NF-κB inhibitors in vitro, which are usually obtained from in vivo observations. These include, for example, delayed onset of hyperglycemia or slowed progression of existing diabetes symptoms. Compounds with this feature are a new class of compounds that can be validated as therapeutic agents for treating these diseases.

In these embodiments, the method comprises selecting test compounds having the characteristics of (i) to (iv) described below, i.e. (i) in vitro as compared to a suitable negative control human or mammalian cell , Compounds that do not activate greater than about 30% of one, two or three of PPAR-α, PPAR-γ and PPAR-δ in human or mammalian cells in vitro; (ii) compounds that inhibit or reduce the transcriptional activity or level of NF-κB in human or mammalian cells by about 20 to 80% in vitro as compared to a suitable negative control human or mammalian cell; (iii) reduced hyperglycemia, slowed the progression or delayed onset of hyperglycemia, increased insulin sensitivity, decreased glucose intolerance, decreased pancreatic β-islet cell number or their insulin as compared to the appropriate negative or normal control group. Slows the rate or progression of the ability to secrete, increases the pancreatic β-islet cell number or their insulin secretion ability, slows the rate of weight gain in db / db mice or feeding induced or obese subjects; , Lowers elevated levels of triglycerides, lowers total blood or plasma cholesterol levels, lowers normal or elevated levels of blood or plasma LDL, VLDL, apoB-100 or apoB-48, or lowers blood or plasma HDL Or increases normal or low levels of apoA1, or elevated levels of fibrinogen in the blood or plasma Chu compound; And (iv) optionally a glucocorticoid receptor, a mineralocorticoid receptor, a progesterone receptor, an androgen receptor, an estrogen receptor-α, an estrogen receptor-β or a human in a mammalian cell, as compared to a suitable negative control human or mammalian cell in vitro. A compound that does not activate greater than about 30% of the activity of one or more of the biologically active variants of the biological molecule. The method allows the identification or characterization of compounds with the potential to treat or alleviate metabolic diseases in humans or other mammals.

In some embodiments, the activity of the test compound can be compared with a suitable reference compound, such as a compound of Formula 1. The compound of formula 1 can be used in the method as a positive control or as a positive reference standard, characterizing the method. Such compounds include 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol and androast-5-ene-3β, 7β, 16α, 17β-tetrol. Other compounds of formula (1) can also be used as negative controls or reference standards that do not have the required characteristics, have one or two or three. Such compounds include 16α-bromoepiandrosterone, 16α-bromo-3β, 17β-dihydroxyandrost-5-ene and 16α-hydroxy piandrosterone.

 Embodiments of the invention also include measuring the effect of a test compound on one or more of the symptoms or symptoms associated with a metabolic disease or disorder. Typically such a measurement is compared to a negative control or a normal control or a suitable positive control, the measurement being performed in vivo in humans or animals, although sometimes such measurements are performed on whole cells or cell lysates in vitro. have.

Hyperglycemic reduction can be observed as a decrease in blood or plasma glucose levels over the normal fasting range (observed as a range of about 70 mg / dL to 105 mg / dL or 115 mg / dL for at least 2 years old humans). The hyperglycemia present at the fasting glucose level is about 135 mg / dL or about 140 mg / dL to 200 mg / dL, 300 mg / dL or 350 mg / dL. Glucose levels higher than about 400 mg / dL can be life threatening. Postprandial blood or plasma glucose levels are measured 2 hours after carbohydrate intake, in humans greater than 75 g when blood is collected for glucose measurement. Post-prandial blood or plasma human glucose levels between 140 mg / dL and 200 mg / dL indicate symptoms of hyperglycemia, and glucose levels above 200 mg / dL indicate human diabetes. In the case of humans, the Oral Glucose Tolerance Test (OGTT) can be performed using blood in patients with normal fasting glucose levels of 70 to 115 mg / dL. In performing OGTT in humans, if the peak glucose level (usually 30 minutes or 1 hour after a meal) and the carbohydrate value more than 200 mg / dL more than two hours later, this indicates that the patient has diabetes will be.

A replacement for blood sugar in humans is a measure of glycosylated hemoglobin or Hb A1c, which is used to monitor diabetes treatment. Measurements of Hb A1c allow blood glucose or glucose levels to be measured 100-120 days prior to testing and are not sensitive to short-term changes such as eating recently or fasting. In adults, Hb A1c levels of 2.2 to 48% are normal, but 2.5 to 5.9% are good controls for diabetes, 6 to 8% are fair to diabetic controls, and levels above 8% are associated with diabetes symptoms. It indicates that the control is not good. How to do this and how to interpret the results and related protocols are described, for example, in KD Pagana and TJ Pagana, Mosby's Diagnostic and Laboratory Test Reference, 5th edition, 2001, Mosby Inc., pages 441-448, 451-458. , 507-509. Treatment with the compound of Formula 1 can also be used to reduce Hb A1c, which is associated with improved diabetes control or treatment.

Using the methods described herein can lead to normalization, such as glucose, glucose replacement levels or other values such as, for example, levels of reactive protein or lipid components over time such as total cholesterol. Means returning to the normal range of the said numerical value or the range of normal vicinity. Normalization of glucose or glucose replacement levels usually results in elevated glucose or glucose replacement levels of about 1%, about 2%, about 3% or about 5% of normal glucose levels, or about 5% or about normal glucose replacement levels. It is observed to fall to the 8% range. Glucose levels for other species are also described, and similar measurements or assays may be used in the present invention instead of such species. Normalization of other levels is usually an abnormally high or low level of regression in the range of about 2% or about 4% to about 6%, about 10%, or about 12% of the upper or lower limit of the normal range of values for the subject species. Is observed.

Compounds identified by the methods of the invention can be used to delay the onset of hyperglycemia, slow its progression, or increase insulin sensitivity to insulin resistance that is expected to be present or reasonably developed. Other effects of the compound include reduced glucose intolerance, slowing the rate or progression of pancreatic β-islet cell number loss, or slowing the rate or progression of the loss of pancreatic β-islet cell ability to secrete insulin. Or an increase in the number of β islet cells or an increase in the ability of the β islet cells to secrete insulin.

In some embodiments, this method may be performed in an obese subject. The term obesity or “overweight” as used in the present invention in humans means that (1) the adult body fat index is about 26 kg / m ² , 27 kg / m ² , 28 kg / m ² , 29 kg / m ² , 30 kg / m ² , 31 kg / m ² , 32 kg / m ² or more, and for adult women the body fat index is at least about 26 kg / m ² , 27 kg / m ² , 28 kg / m ² , 29 kg / m ² , 30 kg / m ² , 31 kg / m ² , 32 kg / m ² or more, or (2) A person who has been assessed as obese or overweight by a health care professional, such as a doctor or nurse. Used. For example, in the measurement of obesity in humans, body fat content and its distribution can be used in the calculations, because some people with high body arrest water often have large amounts of muscle tissue instead of fat or fat cell tissue. This is because a large amount of body fat in the body other than the abdomen or the abdomen, for example, the hips and pelvis, may not be technically determined as obese. Regarding obesity and preservation water, see, eg, GA Colditz, Med. Sci. Sports Exerc ., 31 (11), Suppl., Pp. S663-S667, 1999, FJ Nieto-Garcia et al., Epidemiology , 1 (2): 146-152, 1990, RH Eckel, Circulation , 96: 3248-3250, 1999).

In some embodiments, the compounds identified by the methods of the present invention, when compared to a suitable negative control human or mammalian cell, usually measured in an in vitro assay, may be used in mineral corticoid receptors in human or mammalian cells, At least one of the progesterone receptor, the glucocorticoid receptor, the androgen receptor estrogen receptor-α, the estrogen receptor-β, or a biologically active variant of these biomolecules is significantly greater than about 10%, about 20%, or about 30% Do not activate it. Methods for measuring these activities are described, for example, in US Pat. No. 5,298,429. As one exemplary method, assays for assessing whether a test compound is a functional ligand for a hormone receptor protein, or whether it is a functionally engineered or modified form thereof, include: (a) Hormone receptors Culturing cells containing non-endogenous DNA expressing a protein, or a functionally engineered or modified form thereof, and a DNA encoding an operative hormone receptor factor linked to the reporter gene, wherein the culturing is Performing in the presence of at least one test compound that functions as a ligand or modulator for a hormone receptor protein or a functionally engineered or modified form thereof, and (b) provides evidence that said reporter gene has been transcribed in said cell. Analytical step to discover. This assay will usually be performed using mammalian cells, such as CV-1 or COS cells. The reporter gene may be contained in a reporter plasmid, wherein non-endogenous DNA expressing a hormone receptor protein or a functionally modified form thereof is contained in an expression plasmid, wherein the reporter and expression plasmid are also cloned of SV-40. It may contain an origin. In addition, the reporter gene may be contained in the reporter plasmid, wherein non-endogenous DNA, ie, DNA expressing a hormone receptor protein or a functionally modified form thereof, is contained in an expression plasmid, wherein the reporter and expression plasmid are selected markers. Includes too. Related assays can also use cells that have been stably transfected using detectable reporter genes, for example estrogen receptor-β (ERb-UAS-bla GripTite cell-based Assay, Catalog Number K1091, Invitrogen Corp.), Estrogen receptor-α (ERa-UAS-bla GripTite 293 cell-based Assay Catalog Number K1090, Invitrogen Corp.), androgen receptor (AR-UAS-bla GripTite 293 MSR cell-based Assay, Catalog Number K1082, Invitrogen Corp.) or Progesterone receptor (Progesterone Receptor-UAS-bla HEK293T Assay, Catalog Number K1103, Invitrogen Corp.).

Embodiments of the invention include methods for identifying or characterizing the biological activity of a compound having the potential to treat or alleviate metabolic diseases in a mammal, the method comprising (i) a suitable negative control in vitro Compounds that do not activate more than about 30% of one, two or three of PPAR-α, PPAR-γ and PPAR-δ in vitro in human or mammalian cells compared to human or mammalian cells; (ii) compounds that inhibit or reduce the transcriptional activity or level of NF-κB in human or mammalian cells by about 20 to 80% in vitro as compared to a suitable negative control human or mammalian cell; (iii) reduced hyperglycemia, slowed the progression or delayed onset of hyperglycemia, increased insulin sensitivity, decreased glucose intolerance, decreased pancreatic β-islet cell number or their insulin as compared to the appropriate negative or normal control group. Slows the rate or progression of the ability to secrete, increases the pancreatic β-islet cell number or their insulin secretion ability, slows the rate of weight gain in db / db mice or feeding induced or obese subjects , Lowers elevated levels of triglycerides, lowers total blood or plasma cholesterol levels, lowers normal or elevated levels of blood or plasma LDL, VLDL, apoB-100 or apoB-48, or lowers blood or plasma HDL Or increases normal or low levels of apoA1, or elevated levels of fibrinogen in the blood or plasma Chu compound; And (iv) optionally a glucocorticoid receptor, a mineralocorticoid receptor, a progesterone receptor, an androgen receptor, an estrogen receptor-α, an estrogen receptor-β or a human in a mammalian cell, as compared to a suitable negative control human or mammalian cell in vitro. Compounds that do not activate more than about 30% of the activity of at least one of the biologically active variants of the biological molecule; (v) optionally phosphoenolpyruvate carboxykinase (PEPCK) or 11β-hydroxysteroid dehydrogen in hepatocytes or liver-derived cells or in vivo in hepatocytes or tissues obtained from hepatocytes or hepatocytes. Compounds that inhibit the activity or level of genease (11β-HSD) and optionally inhibit the activity or level of type 1 11β-HSD or type 2 11β-HSD or PEPCK or the level of mRNA encoding 11β-HSD Selecting a step. This method allows the compounds of the present invention to be identified or characterized as having the potential to treat or alleviate metabolic diseases in humans or other mammals. PEPCK enzymes may be present in the cytoplasm or mitochondria of the origin.

Bone loss symptoms

Compositions containing a compound of Formula 1 and one or more excipients include bone loss or osteopenia described herein, such as primary osteoporosis, postmenopausal or type 1 osteoporosis, menopausal or type 2 osteoporosis, idiopathic osteoporosis, Slowing the start to treat and prevent osteoporosis, such as secondary osteoporosis, such as glucocorticoid-related bone loss, and bone loss, associated with trauma, such as thermal, chemical, or radiation burns at 1, 2, and 3 degrees Can be used to slow down or slow down the process. Treatment with the compound of formula 1 can improve bone mass, bone density and / or bone strength.

Drug products

In some embodiments, the present invention provides a drug product for treating inflammatory symptoms. Drug products usually comprise (a) a drug in a dosage form such as a solid or liquid formulation suitable for oral or parenteral administration. The packaging and / or insert or label packaging for the drug may include the efficacy of the drug, the mechanism of action, the target patient population, the dosage, the dosage regimen, the route of administration, the toxicity due to biological injury, and the injury the drug can treat, if known. Information about the severity of If the biological injury is exposure to radiation, the insert or label package may contain information about the dosage of radiation and the range of dosages for which the drug product can be used or approved. The drug product may also optionally contain a diary or instructions for use to record information about when or how the drug is used or about symptoms or drug effects that a drug user may experience during or after using the drug. It can be used to help with stage IV or post-marketing analysis of the efficacy or side effects of the drug. Other embodiments of the drug product are as described in other embodiments herein.

The term drug product, as used herein, refers to a regulatory body that reviews or approves for marketing or medical use, such as the US Food and Drug Administration or the European Medicines Agency or European Medicines. Evaluation Agency) means a drug that has been reviewed and approved by the Agency. The use of drug products includes commercially available and commercially available and purchased for contemplated use. Such activities would normally have to be in line with regulatory policies that could govern the marketing, sale, purchase or drug handling. The drugs in the drug product may be new drugs, generic drugs, biological drugs, medical devices, or protocols for using them. Drug products are usually approved for marketing by the US Food and Drug Administration or European Medicines Agency for applications such as new drug applications, shortened new drug applications, biologically licensed applications, or commercially available pharmaceutical devices. Use of the drug product may be for public or private purchasers, such as the US Department of Defense, the US Department of Energy, the US Department of Health and Human Services, or individual drug buyers or sellers. Includes selling on. Other uses include the use of the drug to treat specified or approved medical conditions, and the approval of a physician for use not indicated on the label. Pre-approval drug products are another aspect of the present invention, which may be essentially the same as the drugs described herein, but may be used to prepare a drug for regulatory review purposes or for commercial use before being approved for commercial use. Say.

The intended patient population identified by the drug product may indicate a population that is excluded if applicable to a pediatric population, an elderly population, or the like. The dosage information usually specifies the daily dose of the drug, but the dosage regimen will explain how often and for how long the drug should be administered. The route of administration represents one or more routes suitable for using the drug, but certain formulations are usually approved for only one route of administration. The dosage, dosage regimen and route of administration written on the packaging may also be described in other parts of the invention.

In one embodiment, the drug product is for the treatment, prevention or alleviation of inflammatory symptoms or other symptoms described herein, which are intended for oral or parenteral administration, for example intramuscular, intradermal or subcutaneous injection. Formulations containing a compound, such as a compound of formula 1, formulated with suitable one, two, three, four or more excipients, the package may have an insert or label describing the daily dosage For example, one, two, three, four, five, six, seven, eight, nine, ten or more consecutive days beginning on the day when the disease or symptom was diagnosed or otherwise the disease was observed. Compounds of Formula 1 over 0.5 days were 0.5 mg, 1 mg, 4 mg, 5 mg, 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 300 Explain that the daily dosage of mg, 350 mg, 400 mg, 450 mg or 500 mg is administered. Will. Information that the package insert or label may contain may include information about the biological response to the drug or the treatment regimen. This information includes (a) a description of one or more adverse or toxic reactions associated with use in mammals, such as humans or non-human primates, (b) effects on inflammatory or other symptoms, (c) dexamethasone or other Protocols or guidelines for using other therapeutic agents, such as glucocorticoids, and (d) time or duration information as to when drug administration should begin for known or best therapeutic uses.

In some aspects, the present invention provides for the number or activity of CD4 ⁺ CD25 ⁺ regulatory T cells, CD4 ⁺ CD25 ⁺ CD103 ⁺ regulatory T cells, CD4 ⁺ CD25 ^high CD103 ⁺ regulatory T cells or CD4 ⁺ CD25 ^high regulatory T cells in a mammal. A method is provided for identifying a compound having the potential to detectably modulate glucocorticoid receptor in human or mammalian cells in vitro as compared to (i) a suitable control human or mammalian cell in vitro. , Compounds that do not activate or inhibit at least about 30% of androgen receptor estrogen receptor-α, estrogen receptor-β or biologically active variants of these biomolecules; (ii) a compound having a molecular weight of about 100 to 1000 Da, optionally a compound having a molecular weight of about 250 to 850 Da; (iii) the number of CD4 ⁺ CD25 ⁺ regulatory T cells, CD4 ⁺ CD25 ⁺ CD103 ⁺ regulatory T cells, CD4 ⁺ CD25 ^high CD103 ⁺ regulatory T cells or CD4 ⁺ CD25 ^high regulatory T cells compared to the appropriate negative control or normal control Or increase or decrease the activity by at least 20% in a suitable assay; And (iv) optionally inhibiting or reducing the transcriptional activity or level of NF-κB in human or mammalian cells by at least about 20-80% in vitro as compared to a suitable negative control human or mammalian cell in vitro. Selecting a step. Compounds of Formula 1 and other compounds may be used in these embodiments as substantially described in Examples 20 or 21 below.

Compounds that increase or decrease the number or activity of specific T cell subsets, such as CD4 ⁺ CD25 ⁺ T cells and CD4 ⁺ CD25 ^high T cells, in vitro or in vivo include autoimmune symptoms, cancer, neurological trauma or ischemia, and the like. To treat the host's symptoms for the graft in conditions such as post-traumatic neuron loss, metabolic diseases such as type 1 diabetes, atherosclerosis, organ or tissue rejection in autologous grafts, and where these symptoms may be present and may occur. Candidates for use in or to slow the start or slow the progression. Treatment can be used to improve wound healing, treat wounds due to reperfusion, stenosis, restenosis after angiography, myocardial infarction or cerebral infarction. Embodiments include compounds having a molecular weight of about 2000 Da or less, compounds of about 1000 Da or less, or compounds of about 500 Da or less. Some compounds have a molecular weight of about 285 or 290 to about 500 or 650 Da. The Treg cell response is compared in the number of Treg cells, typically CD4 ⁺ CD25 ⁺ T cells or CD4 ⁺ CD25 ^high T cells, in a subject treated with the compound, or in an in vitro assay, or in the subject as compared to a suitable negative control. About 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80 %, About 100%, about 200%, about 400%, about 600%, about 1000%, about 2000%, about 5000%, about 10000% or more. Such rates of change can be observed as Treg cell numbers in the blood or cells in vitro or in vivo, or as the rate of increase or decrease in their activity.

Methods for analyzing subset cell profiles, such as T cell profiles, can be obtained by various methods, including flow cytometry (FACS), such as Levy et al., Clin. Immunol. Immunopathol . 35: 328, 1985. In FACS analysis, there are monoclonal antibodies against various subsets of cells for binding to and identifying phenotypic surface antigens presented on cells. There are commercially available antibodies in which the presence of such markers is generally not required since the presence of such markers can be detected. Antibodies that identify identical or highly related antigen markers are expected to produce similar diagnostic results. Therefore, where marker antigens are defined in the specification and claims with reference to, for example, specific monoclonal antibodies that bind to CD4 or CD25, these definitions also include markers when different monoclonal antibodies are used for such identification. It is to include. Corresponding phenotypic markers include CD3 for total T cells, CD4 for T helper / inducer cells, CD8 for T suppressor / cytotoxic cells, and CD16 / 56 for NK cells, and for T suppressor cells CD8-expressing subset markers such as CD11β, CD38 for activated T suppressor / cytotoxic cells, activated T suppressor / cytotoxic cells, and HLA-DR for CD57; And activated T helper / inducer cells, including Treg cells, have CD4 expression markers such as CD25 and HLA-DR.

Dosing protocol or method

In treating the symptoms or symptoms described herein, the compound of formula 1 may be administered continuously (daily) or intermittently to a subject vulnerable to or suffering from said symptoms or symptoms. In treating symptoms, such as inflammatory symptoms or other symptoms described herein, with a compound of Formula 1, intermittent administration may avoid or alleviate some of the undesirable aspects usually associated with discontinuous administration. . In this undesirable aspect, there are undesirable side effects or toxicity, such as preventing daily dosing regimens or reducing the administration of other therapeutic agents such as glucocorticoids and / or bone loss or bone resorption.

In some embodiments, daily administration can be continued as long as the disease or symptoms are evident, typically as long as they are chronic. In other embodiments, daily administration can be continued for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days in succession, leading to a period of time during which no administration is required or administration is needed again. Can be. Such an embodiment generally includes treating acute symptoms that may or may not relapse over time. Treatment of chronic symptoms usually involves continuous administration for an extended period of time.

In the treatment of continuous (daily) or intermittent dosing regimens, or the diseases, symptoms or symptoms described herein, the compounds of formula (I) may be used in one or more suitable routes, for example oral, buccal, sublingual, topical, It can be administered intramuscularly, intracutaneously, subcutaneously, intravenously, skin or as an aerosol.

Dosage is usually from about 0.05 mg / kg / day to about 200 mg / kg / day. Typical dosage ranges are from about 0.1 to about 100 mg / kg / day, including about 0.2 mg / kg / day, 0.5 mg / kg / day, about 1 mg / kg / day, and about 2 mg. / kg / day, about 4 mg / kg / day, about 5 mg / kg / day, about 6 mg / kg / day, about 8 mg / kg / day, about 10 mg / kg / day , About 20 mg / kg / day, about 40 mg / kg / day or about 100 mg / kg / day. Higher doses such as about 250 mg / kg / day, about 300 mg / kg / day or about 350 mg / kg / day may also be used, for example, in vertebrates. The compound of formula 1 may be administered orally or parenterally, about 2 to about 50 mg / kg / day or about 2 to 40 mg / kg / day. Such administration generally results in a plasma level of the compound of Formula 1 being from about 4 ng / mL or from about 8 ng / mL to about 125 ng / mL or about 250 ng / mL, for example from about 15 ng / mL to about 120 ng / mL or about 20 ng / mL to about 100 ng / mL. Such plasma levels may be transient, for example, as may occur or after administration of the compound for a depot formulation, and may last from about 30 minutes or about 60 minutes to about 2 hours or about 8 hours. .

Continuous daily administration is usually used to treat the chronic symptoms described herein. Daily administration is usually given in a single dose, but even daily doses may be divided into two to three subunit doses. An intermittent dosing protocol includes administering a compound of Formula 1 every other day or once every three days for an appropriate period of time. In the case of treating erythrocyte deficiency, administration usually begins on the same day that the subject experiences a short-term myeloablative event such as radiation exposure. In long-term cases, for example for chemotherapy due to cancer, the administration of Formula 1 is about 12 hours, about 1 day, about 2 days, or about 3 days after the chemotherapeutic agent is administered to the subject. You can start Daily administration can last for a specified period of time, followed by a period of non-administration on a fixed or alternative time period. In this embodiment, disease flares, such as multiple sclerosis, arthritis, asthma, colitis or Crohn's disease, are administered daily for 3 to 14 consecutive days or 5 to 10 consecutive days, followed by another recurrence or It can be treated by not treating it until it starts.

Clinical Symptoms and Symptoms

The compounds and methods described herein are useful for treating, alleviating, preventing, or slowing the progression of the symptoms and / or one or more of the symptoms described herein. Such uses include inhibiting bone resorption, reducing undesirable side effects associated with or recurring by chemotherapeutic agents such as anti-inflammatory glucocorticoids, treating, preventing or slowing osteoporosis and fractures, Inhibiting vascular restenosis, and retinopathy, spot degeneration, inflammation, undesirable inflammatory symptoms or symptoms due to diabetes, such as pulmonary inflammatory symptoms such as fibrocystic cysts, acute or chronic asthma, bronchial asthma, atopy Asthma, ARDS or COPD, or autoimmune diseases such as osteoarthritis, rheumatoid arthritis, pancreatitis such as autoimmune pancreatitis, systemic lupus erythematosus, lupus erythema related tissue inflammation, lupus erythema related arthritis, lupus erythema related skin changes, lupus erythema related Abnormal blood, lupus erythema-related kidney damage, lupus erythema-related heart or lung disease, and It includes undesirable lupus erythematosus-related psychiatric care or treatment of neurologic changes.

Symptoms that can be treated include fever, joint pain (joint pain), arthritis, and meningitis (pleurisy or pericarditis). Administration of other agents can also be used in the treatment of the present invention. Therefore, pain can be treated with nonsteroidal anti-inflammatory agents such as aspirin, salicylate, ibuprofen, naproxen, clinolyl, oxaprozin and tolmetin. The skin of systemic lupus can be treated with antimalarial drugs such as hydroxychloroquine, chloroquine and quinacrine and the like. Retinoids such as istretinoin and etretinate, and the like, can also be used to treat skin symptoms in combination with the compounds described herein. Organ damage can be treated with corticosteroids, which are usually administered orally or intravenously. Corticosteroids that can be used include hydrocortisone (crotisol), corticosterone, aldosterone, ACTH, triamcinolone and triamcinolone diacetate, derivatives thereof such as triamcinolone hexacetonide and triamcinolone acetonide, betamethasone and betamethasone dipropionone Derivatives thereof, such as nates, betamethasone benzoate, betamethasone sodium phosphate, betamethasone acetate and betamethasone valerate, flunisolide, prednisone and derivatives thereof, fluocinolone and fluorinone acetonide Fluocinolone derivatives, such as diflorasone and diflorasone diacetate, and derivatives thereof such as halcinonide, dexamethasone and dexamethasone dipropionate and dexamethasone valerate Derivatives such as conductors, desoxymetasone (desoxymethasone), diflucortolone and diflucortolone valerate, fluclorolone acetonide, fluorinolone Derivatives thereof, such as fluorocinonide, fluocortolone, fluprednidene, flurandrenolide, clobetasol, clobetasone and clobetason butyrate, Alclometasone, flumethasone and fluorocortolone.

In cases where oral administration of corticosteroids is insufficient, intravenous methyl prednisone pulse therapy (high doses) may be used, including lupus nephritis, anemia, central nervous system inflammation (encephalitis), low platelet counts and severe pleural pericarditis, etc. Can treat symptoms other than severe kidneys.

Compounds of formula (1) can be used to treat, prevent or slow the progression of osteoporosis or fractures. Treatment of the subject may lead to bone strength strengthening and / or reduced loss of bone mass or minerals, thereby increasing resistance to fracture. As used herein, the term "treatment" of a symptom means that such treatment can alleviate, prevent or slow the progression of such symptoms and / or alleviate or prevent one or more symptoms of such symptoms, or That means you can slow it down.

The compound of formula 1 may be used to treat metabolic diseases such as type 1 diabetes, type 2 diabetes, hyperglycemia, insulin resistance, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, lipotrophy, X syndrome and atherosclerosis. It can be used to treat, prevent, slow the progression, or delay the onset.

Formulations and Compositions for Formulation Preparation

Embodiments of the invention include this portion and the formulations described elsewhere herein. It is also possible to administer the compound of formula 1 alone, but it is usually present as a formulation. Vertebrate and human formulations comprise at least one excipient and optionally at least one additional therapeutic ingredient, together with at least the compound of formula (I).

Formulations include compositions comprising one, two, three, four or more pharmaceutically acceptable excipients or carriers. The composition can be used to prepare formulations suitable for human or animal use. Suitable routes of administration for formulation include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, anal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intramedullary, intraocular and extramedullary). . Typically aqueous or non-aqueous liquid or cream formulations are delivered via parenteral, oral or topical routes. In one embodiment, as described in the intermittent method of administration of the present invention, the compound of formula 1 may be administered by the route of administration described herein, for example oral, topical, buccal, sublingual, parenteral, inhalation aerosol, or It may be formulated as an aqueous or non-aqueous liquid formulation or as a solid formulation, suitable for routes such as subcutaneous reservoirs, parenteral or intramuscular reservoirs and the like. It will be appreciated that the preferred route of administration may vary depending on, for example, the subject's pathological condition or the subject's body weight or other treatments suitable for the subject's response to the treatment with a compound of Formula 1 or the environment.

Formulations include those suitable for the route of administration described above. The formulations may conveniently be presented in unit dosage form and may be prepared by methods known in the pharmaceutical art. Techniques, excipients and formulations are generally described, for example, in Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, PA 1985, 17 ^th edition, Nema et al., PDA J. Pharm. Sci. Tech . 1997 51: 166-171, G. Cole, et al., Editors, Pharmaceutical Coating Technology , 1995, Taylor & Francis, ISBN 0 136628915, HA Lieberman, et al., Editors, Pharmaceutical Dosage Forms , 1992 2 ^nd revised edition, volumes 1 and 2, Marcel Dekker, ISBN 0824793870, JT Carstensen. Pharmaceutical Preformulation , 1998, pages 1-306, Technomic Publishing Co. It is described in ISBN 1566766907. Exemplary excipients for the formulation include emulsifying wax, propyl gallate, citric acid, lactic acid, polysorbate 80, sodium chloride, isopropyl palmitate, glycerin, white kerosene and other excipients described herein.

Compositions for use in preparing the formulations described herein, or formulations suitable for administration by the routes described herein, optionally have an average particle size of about 0.01 to about 500 microns, about 0.1 to about 100 Particles that are microns or about 0.5 to about 75 microns particles. Average particle sizes range from 0.01 and 500 microns, 0.05 microns or 0.1 microns or other sizes, for example average particle sizes of about 0.05, 0.1, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 , 5.5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 85, 100, 120, and the like. When using a compound of formula (1) or a composition comprising a compound of formula (1), since compounds of formula (1) are used as intermediates for preparing formulations, they have an average particle size or size range of 1, 2, 3, or It may include more than that. In preparing a composition or formulation comprising a compound of Formula 1 (optionally one or more excipients) described herein, optionally a mill for granulating the compound or composition to obtain the desired particle size ), Sieves, or other instruments.

In some embodiments, the compound of formula 1 used is characterized by a lack of appreciable androgencity. In this embodiment, the compound of formula (I) is a compound of androgens such as testosterone, testosterone propionate, dihydrotestosterone or dihydrotestosterone propionate, as determined by a suitable assay using an appropriate positive and / or negative control. Androgen levels are characterized by about 30% or less, about 20% or less, about 10% or less, or about 5% or less. Suitable assay methods for androgen levels of various compounds are described, for example, in JR Brooks, et al., Prostate 1991, 18: 215-227, M. Gerrity et al., Int. J. Androl. 1981 4: 494-504, SS Rao et al., Indian J. Exp. Biol. 1969 7: 20-22, O. Sunami et al., J. Toxicol. Sci. 2000 25: 403-415, GH Deckers et al., J. Steroid Biochem. Mol. Biol. 2000 74: 83-92. The androgen numerical properties of the compound of formula 1 are optionally measured as described in this assay or other methods, or as described essentially.

Therefore, one such embodiment includes the conditions described herein, including administering to a subject in need thereof an effective amount of a compound of formula 1, or delivering an effective amount of a compound of formula 1 to a tissue of a subject. A method of treating a compound of formula (I), wherein the compound of formula (I) comprises testosterone, testosterone propionate, dihydrotestosterone or dihydrotestosterone pyriophyte, as determined by a suitable analytical method as described in the Androgen levels of androgens such as tate are less than about 30%, about 20% or less, about 10% or less or about 5% or less. In carrying out this method, the subject or mammal, such as a rodent, human or primate, is optionally monitored whether the disease, symptom or prognosis is for example alleviated, prevented or reduced in severity. Such monitoring may optionally include cytokines (eg, TNFa, IL-13, IL-1b), WBCs, platelets, granulocytes, neutrophils, RBCs, NK cells, as described herein, or as described in the cited literature. Measuring at least one level of nucleated cells of different types of immune cells during a suitable time cycle, including, for example, starting at baseline prior to treatment, and at various time points after treatment with a compound of Formula 1, such as It can be measured in a manner that ends after about 2 to 45 days after treatment with the compound.

As noted above, some embodiments of treatment with the compound of Formula 1 may be combined with the use of corticosteroids or glucocorticoids. Corticosteroids can be used in a number of clinical situations, including acute or chronic rheumatoid arthritis, acute or chronic osteoarthritis, colitis symptoms such as ulcerative colitis, acute or chronic asthma, bronchial asthma, psoriasis, systemic lupus erythematosus, hepatitis, It is used to lower the intensity or frequency of flares or episodes of inflammatory or autoimmune reactions in symptoms such as fibrotic pneumonia, type 1 diabetes, type 2 diabetes or Cacexia. However, many corticosteroids have significant side effects or toxicity that may limit their use or efficacy. Compounds of Formula 1 are useful for attenuating these side effects or toxicity without compromising all of the desired therapeutic ability of corticosteroids. This allows for continuous use of the corticosteroid, modified doses, for example increased doses, without reducing the corticosteroid dose or without toxicity or side effects. Treatment, prevention, alleviation, side effects or toxicity that may be reduced include bone loss, decreased bone growth, improved bone resorption, osteoporosis, immunosuppression, increased susceptibility to infections, mood and personality changes, depression, headache, Dizziness, high blood pressure, muscle weakness, fatigue, vomiting, discomfort, pepsin ulcers, pancreas, thin or torn skin, growth inhibition, thromboembolism, cataracts and eczema in pre-child or adult subjects. Dosage, route of administration, and administration protocol for the compound of Formula 1 may be the same as described essentially herein. As an exemplary dosage of Formula 1, about 0.5 to about 20 mg / kg / day during administration of a corticosteroid, optionally about 1 week to about 6 months or longer after the completion of the corticosteroid administration Is administered. Corticosteroids can be administered essentially using known dosages, routes of administration, and administration protocols, for example in Physicians Desk Reference 54 ^th edition, 2000, pages 323-2781, ISBN 1-56363-330-2 See, Medical Economics Co., Inc., Montvale, NJ. However, the dosage of the corticosteroid can optionally be adjusted to increase from about 10% to about 300% above the normal dosage without, for example, increasing in terms of side effects or toxicity of the corticosteroid. This increase will be appropriately adjusted for a sufficient period of time depending on the clinical condition of the subject, for example, the daily dose of corticosteroid is from about 10% to about 20%, up to about 300, over about 2 weeks to about 1 year. % Can be increased.

The method of treatment can be used to treat, prevent or alleviate acute trauma such as myocardial infarction, bleeding such as cerebral hemorrhage or stroke or fracture, osteoporosis or excessive or undesirable bone resorption or loss. Treatment involves damage or injury to the skin, mucous membranes, cartilage, liver, heart tissue, bone or CNS or nerve tissue, such as chemical or thermal burns, osteoarthritis, rheumatoid arthritis, liver cirrhosis, osteoporosis, osteoporosis, myocardial infarction It can be used to facilitate repair of stroke or cerebral trauma. Treatment also includes bone loss that may result from glucocorticoid treatment in patients with, for example, lupus symptoms or inflammatory bowel disease, Crohn's disease, acute or chronic colitis or kidney disease such as acute or chronic renal failure or autoimmune kidney wounds. It can also be used to reduce.

The following embodiments illustrate one or more aspects of the present invention.

1.Identifying or identifying a biomechanical compound comprising exposing the subject to an acute stimulus or biological injury causing a detectable response to the acute stimulus or biological injury, and then measuring the biological response of the test compound in vivo in the subject. As a method for characterization, the test compound is used to stimulate or bioinjury during (i) the maximum or near maximum of the acute response, or (ii) when the acute response is increased during the extended acute biological response. Inducing a desired therapeutic response to a mediator of an acute biological response to the above, wherein the preferred therapeutic response at time point (i) or (ii) is one, two, three, or earlier or later. At later stages it differs from its effect on the mediator of acute biological responses. This effect at the earlier or later stages described above will have about the level or activity of the mediator or activity of the acute biological response as compared to the appropriate vehicle or placebo control at the same or substantially the same earlier or later stages. Increasing or decreasing to less than 50% results in a desired treatment response for the mediator of an acute biological response, and the preferred treatment response at time point (i) or (ii) is earlier or later in time. Compounds that differ from their effect on the mediator of acute biological responses at one, two, three, or more of the time periods are identified as biomechanical compounds.

2. In Embodiment 1, further comprising carrying out a protocol to determine whether the test compound modulates the activity or level of the mediator of an acute biological response from about 25% to about 75% in an in vitro assay method. Wherein the test compound optionally does not activate or antagonize the glucocorticoid receptor by at least about 20% compared to a suitable reference active agent or antagonist for the glucocorticoid receptor.

3. In embodiments 1 or 2, the acute stimulation or biological injury is exposing the bowel to a sufficient amount of ionizing radiation or pre-inflammatory signal, to a compound or composition, optionally to the above proinflammatory signal, The compound or composition is bacterial LPS or TNFα and / or optionally the mediator of said acute response is NF-κB or IκB.

4. The method of embodiment 1, 2, or 3, wherein the acute stimulus or biological injury is caused by administering a sufficient amount of bacterial LPS to a sufficient number of drugs, mice, or a sufficient number of vehicle control mice, followed by (I) about 1.5 hours after the intraperitoneal injection of bacterial LPS when the acute response is at or near maximum, (ii) administering a sufficient amount of bacterial LPS One or two time points before and / or after, optionally before administering a sufficient amount of bacterial LPS, and at some time after the acute response reaches a maximum or near maximum, optionally intraperitoneally injected with bacterial LPS Measuring about 2.0 or 2.5 hours after administration, wherein optionally the mediator of an acute biological response is NF-κB or IκB.

5. In embodiment 4, the administration of a sufficient amount of bacterial LPS is carried out essentially according to the method described in example 9, or according to an appropriate modification thereof, optionally wherein the compound is selected from a mediator of an acute biological response. The ability to partially adjust the activity or level is performed essentially according to the method described in Example 7, or according to a suitable variant thereof.

6. For embodiments 1, 2, 3, 4, or 5, comprising a positive biomechanical drug control, optionally including 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol Measuring the efficacy or effect of each of the test compounds, optionally including a biostatic drug control.

7. A drug product or pre-approval drug product comprising a dosage form of a drug together with a package insert or label package containing information on the efficacy, mechanism of action, or clinical use of the drug, wherein the effect, mechanism of action or clinical The information on the intended use is at least in part obtained from a characterization method comprising the method of Embodiment 1, 2, 3, 4, or 5.

8. A drug product or pre-approval drug product comprising a dosage form of a drug together with a package insert or label containing information about the drug's efficacy, mechanism of action, or clinical use, wherein said efficacy, mechanism of action or clinical Information on use may include: (a) contacting the cells in vitro for a sufficient time with a sufficient amount of active agent of NF-κB activity, thereby detectably increasing the activity or level of NF-κB in the cell, Being capable of reacting to the active agent; (b) contacting the cells in vitro for a sufficient amount of drug and for a sufficient time period, wherein the drug detectably inhibits the activity of NK-κB activity relative to the appropriate control; And (c) optionally comparing the ability of the reference compound to a drug that inhibits the activity of NF-κB, wherein the reference compound is a compound described herein by Formula 1, wherein the activity of NF-κB in a characterization method Is a compound capable of detectably inhibiting from about 25% to about 75%, wherein the drug inhibits from about 25 to about 75% of the activity of NF-κB in a characterization method, optionally wherein the reference compound or drug is Does not detectably or significantly bind to the glucocorticoid receptor, or optionally the reference compound or drug does not detectably or significantly functionalizes the glucocorticoid receptor, and optionally the drug is about From 20% more glucocorticoid receptors). The drug product is also obtained some.

9. The drug product according to embodiment 7 or 8, wherein the dosage form comprises an oral, parenteral, topical or inhalation formulation.

10. The drug product of embodiment 8 or 9, wherein the reference compound or drug inhibits from about 35% to about 70%, or from about 40% to about 65%, the activity of NF-κB in the characterization method.

11. In embodiments 8, 9 or 10, NF-κB in the cells is TNF-α, TNF-β, TGF-β, IL-1, epithelial growth cells, bacterial LPS, bacterial peptidoglycan, yeast zymoic acid (zymosan), a bacterial lipoprotein, a bacterial or viral antigen or gene product, activated by one or more of ultraviolet irradiation, heat or temperature increase, lymphokine or oxidative free radicals, or H ₂ O ₂ .

12. For embodiments 8, 9, 10 or 11, the reference compound or drug directly binds to the glucocorticoid receptor with> 10 mM k _d in a suitable binding assay wherein the reference compound or drug is a glucocorticoid receptor mediated gene A drug product that does not detectably functionalize the glucocorticoid receptor at a concentration of about 10 μM or more in an assay suitable for detecting activation or increase in expression.

13. In embodiments 8, 9, 10, 11 or 12, the cells in vitro are mammalian, rodent or human cells, optionally human THP-1 cells, rat RAW cells, macrophages, monocytes, T-lymphocytes, The drug product is selected from the group consisting of B-lymphocytes, dendritic cells, glia cells, Kupfer cells, hepatocytes, neutrophils, leukocytes and whole blood.

14. In Embodiments 7 or 8, information regarding the efficacy, mechanism of action, or clinical use of the drug is included in the regulatory or review agency's regulations to review or approve the commercial use or commercial sale of the drug product. Drug products.

15. Inflammatory symptoms or autoimmunity in a mammal, comprising administering to an individual or delivering to a subject's tissue an effective amount of a biomechanical compound identified by the method of Embodiment 1, 2, 3, 4, 5, or 6 As a method of treating symptoms, wherein a positive biomechanical compound is used as a reference standard, wherein the biomechanical compound is one that partially inhibits the mediator of an acute biological response in a suitable in vitro assay, wherein a suitable in vitro assay is optionally an example. Essentially according to the method of 7, or a variant thereof.

16. A purified compound having the structure

Wherein ^one of R ¹ is —H or C _1-8 optionally substituted alkyl and the other R ¹ is —OH, C _2-8 ester or C _1-8 ether; One of R ² is —H or C _1-8 optionally substituted alkyl and the other R ² is —H, —OH, a C _2-8 ester or a C _1-8 ether; One of R ³ is —H or C _1-8 optionally substituted alkyl and the other R ³ is —OH, C _2-8 ester, C _1-8 ether or C _1-8 optionally substituted alkyl Is; One of R ⁴ is —H or C _1-8 optionally substituted alkyl and the other R ⁴ is —OH, C _2-8 ester or C _1-8 ether; R ⁵ is —CH ₃ , —C ₂ H ₅ or —CH ₂ OH; R ⁶ is —H, —CH ₃ , —C ₂ H ₅ or —CH ₂ OH; One of R ⁷ is —H or C _1-8 optionally substituted alkyl and the other R ⁷ is —H, —OH, a C _2-8 ester or a C _1-8 ether; R ¹⁰ is -H or halogen; One of R ¹¹ is —H or C _1-8 optionally substituted alkyl, and the other R ¹¹ is —H, —OH, C _2-8 ester, C _1-8 ether or C _1-8 A compound that is optionally substituted alkyl.

17. Purified compound according to embodiment 16, wherein one, two or three of R ² , R ⁷ or R ¹¹ is —OH, C _2-8 ester, C _1-8 ether, ═O or = NOH .

18. In embodiment 17, the compound comprises 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androst-5-ene-3β, 7β, 16α, 17β-tetrol, 17α Ethynylandrost-5-ene-3β, 7β, 16α, 17β-tetrol, androst-5-ene-3β, 7α, 16α, 17β-tetrol, androst-5-ene-3β, 4β, 16α, 17β-tetrol, androast-5-ene-3α, 4β, 16α, 17β-tetrol, androast-5-ene-3β, 11β, 16α, 17β-tetrol, androast-5-ene- 3α, 11β, 16α, 17β-tetrol, 3β, 11β, 16β, 17β-tetrol, androst-5-ene-3α, 11β, 16β, 17β-tetrol or C _2-4 monoesters of these compounds or C _2-4 diester derivatives, optionally wherein (1) the C _2-4 monoester is acetate or propionate at the 3- or 17-position, or (2) the C _2-4 diester is 3 Or an acetate or propionate at the 17-position.

19. The compound of embodiment 17 or 18, wherein the compound is (a) a powder or granule that is at least 80% pure, at least 95% pure or at least 98% pure, or (b) at least 80% pure, at least 95% pure or at least Purified compound, which is a solution or suspension in 98% purity. Such compounds include 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androst-5-ene-3β, 4β, 16α, 17β-tetrol, androst-5-ene-3β , 11β, 16α, 17β-tetrol, androst-5-ene-3β, 7β, 16α, 17β-tetrol and one or two hydroxyl groups of these compounds are α- to β- or β- to α- Epimers of such compounds that have been replaced with, e.g., 17β-ethynylandrost-5-ene-3β, 7β, 17α-triol, androast-5-ene-3β, 4β, 16α, 17α-tetrol , 17α-ethynylandrost-5-ene-3α, 7β, 17β-triol or androast-5-ene-3α, 4β, 16α, 17β-tetrol.

20. The compound of embodiment 17, 18, or 19, wherein the compound is about 80%, about 85%, about 90%, about 95%, about 97% or about 98% to about 99.5% or about 99.9% pure And optionally the compound is in the form of a powder or granules, and optionally the average particle size of the powder is about 50 nm or about 100 nm to about 5 μm, about 10 μm or about 25 μm, as determined by a suitable assay such as light scattering or the like. Purified compound.

21. Detectable control of the number or activity of CD4 ⁺ CD25 ⁺ regulatory T cells, CD4 ⁺ CD25 ⁺ CD103 ⁺ regulatory T cells, CD4 ⁺ CD25 ^high CD103 ⁺ regulatory T cells or CD4 ⁺ CD25 ^high regulatory T cells in mammals. As a method of identifying compounds having potential,

(i) in glucocorticoid receptors, androgen receptor estrogen receptor-α, estrogen receptor-β or biologically active variants of these biomolecules in human or mammalian cells in vitro as compared to appropriate control human or mammalian cells in vitro. Compounds that do not activate or inhibit one or more of about 30% or more; (ii) a compound having a molecular weight of about 100 to 1000 Da, optionally a compound having a molecular weight of about 250 to 850 Da; (iii) the number of CD4 ⁺ CD25 ⁺ regulatory T cells, CD4 ⁺ CD25 ⁺ CD103 ⁺ regulatory T cells, CD4 ⁺ CD25 ^high CD103 ⁺ regulatory T cells or CD4 ⁺ CD25 ^high regulatory T cells compared to a suitable negative or normal control, or To increase or inhibit the activity by at least 20% in a suitable assay; And (iv) optionally inhibiting or reducing the transcriptional activity or level of NF-κB by at least about 20-80% in human or mammalian cells in vitro as compared to a suitable negative control human or mammalian cell in vitro. A method for identifying a compound, comprising selecting a compound.

22. The method of embodiment 21, wherein the mammal is rodent or human.

23. In either embodiment 21 or 22, the number or activity of CD4 ⁺ CD25 ⁺ regulatory T cells, CD4 ⁺ CD25 ⁺ CD103 ⁺ regulatory T cells, CD4 ⁺ CD25 ^high CD103 ⁺ regulatory T cells or CD4 ⁺ CD25 ^high regulatory T cells (a) the method of example 20 or a suitable variant thereof; (b) the method of example 21 or a suitable variant thereof; Or (c) a method described in the references of the present invention or a suitable modification method thereof, wherein the suitable modification method is a CD4 ⁺ CD25 ⁺ regulatory T cell, CD4 ⁺ CD25 ⁺ CD103 ⁺ regulatory T cell, CD4 ⁺ CD25 ^high CD103 ⁺ regulatory T Measuring by a protocol comprising one, two or three of the methods to allow measurement of the number or activity of cells or CD4 ⁺ CD25 ^high regulatory T cells.

24. The embodiment 21, 22, or 23, wherein the compound is for the treatment or prevention of autoimmune diseases or undesirable inflammatory symptoms, optionally osteoarthritis (primary or secondary osteoarthritis), rheumatoid arthritis , Lupus symptoms such as osteoarthritis, polysclerosis, Alzheimer's disease, hay salt, erythematous lupus or disk-like lupus erythematosus associated with spondylitis such as ankylosing spondylitis, tendonitis, bursitis, asthma, emphysema, chronic obstructive pulmonary disease And pulmonary inflammatory symptoms such as cystic fibrosis, acute or adult respiratory distress syndrome, chronic bronchitis, acute bronchitis, bronchitis, bronchial fibrotic obstruction, bronchial obstruction associated with formation pneumonia. The compound may be a compound of Formula 1 as described herein.

Modifications of the present embodiment and other parts of this specification will be apparent to those skilled in the art upon reading this specification. Such modifications also belong to the scope of the present invention. References cited in the present invention are also incorporated herein by reference at designated locations or in other paragraphs following the paragraph.

The following examples further illustrate the invention and do not limit the invention in any way.

Example 1

Treatment of pulmonary inflammation. Three compounds, 3β, 16α-dihydroxy-17-oxoandrostane, 3α, 16β, 17β-trihydroxyandrostane and 3α, 16α, 17α-trihydroxyandrostane, are described in D. Auci et. al., Ann.New York Acad. Sci . 1051: 730-742 2005, newly cited), to treat inflammation in mice. CD1 male mice from 5 to 8 weeks old (Charles River, Calco, Italy) were used in this study. Experimental animals were housed under controlled environment and fed standard rodent food and water. Animal care was carried out in compliance with regulations on animal care. Mice were assigned to one of the following groups: (1) mice treated with 2% carageenan-λ in saline (carrageenan-λ treated control), (2) carrageenan-λ administration Mice treated with subcutaneous (sc) injections of 0.1 mg, 0.01 mg or 0.001 mg of 3β, 16α-dihydroxy-17-oxoandrostan 24 hours and 1 hour before, (3) 24 and 1 hour prior to carrageenan administration, Mice treated with subcutaneous injection of 0.1 mg, 0.01 mg or 0.001 mg of 3α, 16α, 17α-trihydroxyandrostan, (4) 0.1 mg, 0.01 mg or 0.001 mg 3α, 24 hours and 1 hour before carrageenan-λ administration Mice treated with subcutaneous injection of, 16β, 17β-trihydroxyandrostane, (5) 24 hours and 1 hour before carrageenan-λ administration, vehicle (0.1% carboxymethylcellulose, 0.9% saline, 2% tween 80, Mice treated with subcutaneous injection of 0.05% phenol), (6) 24 h and 1 h before carrageenan-λ administration Mice treated with rabbit anti-mouse polyclonal anti-TNF-α antibody (200 μg) as intraperitoneal bolus (positive control), and (7) sham-operated not treated with carrageenan-λ mice). Each group consists of 10 mice. All treatments were made to 100 μl final volume. Pulmonary (muscle) infection was caused by: Mice were anesthetized with isoflurane and skin incisions were made at the left sixth rib location. Muscles underneath were removed and injected into the rib with 0.1 ml saline (control) or 0.1 ml saline containing 2% λ-carrageenan. Carrageenan-λ can cause inflammation, which is caused by the accumulation of fluid and the accumulation of intraneutrophil neutrophils in this protocol. The incision is closed with a suture and the animal is restored.

Four hours after injection of carrageenan-λ, animals were euthanized by exposing CO ₂ to the animals. The chest was carefully opened and the ribs were washed with 1 ml saline containing heparin (5 U / ml) and indomethacin (10 μg / ml). The effluent and washings were aspirated off and the total volume was measured. Effluent containing blood was discarded. The amount of effluent was calculated by subtracting 1 ml of the injected volume from the total rib volume recovered. Neutrophils in the effluent were suspended in phosphate buffered saline and counted by optical microscopy in a Bucker's chamber after trypan blue staining. The results were analyzed by one-way ANOVA and Bonferroni post-hoc test for multi - sided comparison. P-values less than 0.05 were considered significant. For statistical analysis, each group was compared to carrageenan-λ challenged mice and other untreated control mice.

All of the mice challenged with carrageenan-λ were untreated with acute pleurisy, had increased neutrophil counts and showed cloudy effluents. Elevated effluent volume and leukocyte counts were increased in the ribs of the vehicle treated mice, similar to control mice challenged with carrageenan-λ and untreated control mice. For two groups of control mice, animals treated with 3β, 16α-dihydroxy-17-oxoandrostane had a significant reduction in neutrophil effluent volume and neutrophil counts in the effluent at 0.1 mg, 0.01 mg doses. In contrast, the lower 0.001 mg dose showed no activity. In the mice administered the 0.1 mg dose of 3β, 16α-dihydroxy-17-oxoandrostane, the rib effusion was significantly reduced, but not at the 0.01 mg and 0.001 mg doses. Animals treated with 3α, 16α, 17α-trihydroxyandrostane showed a significant decrease in intramuscular neutrophil counts at 0.1 mg and 0.01 mg doses. Animals treated with 3α, 16β, 17β-trihydroxyandrostane showed a significant decrease in intraneuronal neutrophil counts at 0.1 mg and 0.01 mg doses. The efficacy of 3α, 16α, 17α-trihydroxyandrostan and 3α, 16β, 17α-trihydroxyandrostan was similar to that observed in the polyclonal anti-TNF-α antibody control, but 3β, 16α-dihydroxy -17-oxoandrostan was lower.

The table below describes the neutrophil counts from treated animal groups compared to untreated control animals (negative controls) that were exposed to carrageenan-λ but not to others. The neutrophil count for the negative control was 100% and the other group was compared to this value. The animal group treated with anti-TNF-α antibody (positive control) was 29% of the neutrophil number of the negative control, indicating that the antibody has an anti-inflammatory effect on carrageenan-λ exposure. The vehicle control group did not significantly reduce neutrophil counts compared to the negative control group (91%), indicating that there was no significant anti-inflammatory effect due to vehicle alone.

3β, 16α-dihydroxy-17-oxoandrostane 3α, 16α, 17α-trihydroxyandrostan 3α, 16β, 17β-trihydroxyanthrostane 0.001 mg 97% 0.001 mg 103% 0.001 mg 95% 0.01 mg 73% 0.01 mg 45% 0.01 mg 50% 0.1 mg 73% 0.1 mg 30% 0.1 mg 42%

Other compounds with statistically significant anti-inflammatory activity in this model are 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol (administered via 1 mg and 0.1 mg oral feeding) and 17β-amino Androast-5-ene-3β-ol (40 mg / kg, administered via oral feeding as about 0.5 mg / mouse). These compounds were more active than the group of mice used as vehicle controls.

Other compounds of formula (I) described herein may also be used to characterize, respectively, the ability to treat or alleviate inflammation in this manner. Such compounds include 3β, 16β, 17β-trihydroxyandrostane, 3β, 16α, 17α-trihydroxyandrostane, 3β, 16β, 17α-trihydroxyandrostane, 3β, 16β-dihydroxyandrost- 5-ene-17-oxime, 3β, 16α-dihydroxyandrost-5-ene-17-oxime, 3α, 16α-dihydroxyandrost-5-ene-17-oxime, 3β, 16α-dihydrate As loxyandrostan-17-oxime and analogs of these compounds, (1) a compound containing a hydroxyl group at position 7 in the α or β conformation and / or (2) at the 5- or 4-position Compounds containing double bonds and / or (3) analogs which are esters, ethers, amino acids, carbamates or oxime (= NOH) derivatives, conjugates or analogs of these compounds.

Example 2

Analysis of the immune response. It was determined whether the compounds 3α, 16α, 17α-trihydroxyandrostane have biological properties that make the compounds superior as therapeutic agents for treating inflammatory reactions such as asthma. Specifically, the use of this compound did not involve rebound in IL-13, which is one of the known side effects of anti-inflammatory glucocorticoids such as dexamethasone. IL-13 rebound after glucocorticoid administration makes asthma patients susceptible to future acute relapses, so anti-inflammatory agents that do not have these properties would be advantageous. It is not obvious that IL-13 rebound does not occur.

Ovalbumin (OVA) limits the ability of 3α, 16α, 17α-trihydroxyandrostane to limit eosinophil burden and reduce key inflammatory mediators (L-5, IL-13, cysteinly leukotrienes) Observed in a mouse model of sensitized asthma. BALB / c mice were sensitized by injection of OVA (in alum adjuvant) intraperitoneally on Day 1 and Day 12. On day 28 and 30 after delivery of the OVA to the lungs, the airways were challenged with OVA or challenged with saline. At day 31, the mice challenged with saline in 6 mice and OVA in 6 mice were then sacrificed for lung tissue. The remaining animals were divided into six groups (6 per group). Mice groups were treated with subcutaneous injections once a day as follows. Group 1 vehicle control (0.1% carboxymethyl cellulose, 0.9% saline, 2% tween 80, 0.05% phenol). Group 2 dexamethasone (5 mg / kg). Group 3 3α, 16α, 17α-trihydroxyandrostane (1 mg / mouse). Three animals in groups 1-3 were sacrificed on day 35, 1 hour after the last treatment, and three animals in the remaining 1-3 groups were sacrificed on day 38.

As shown in the table below, 3α, 16α, 17α-trihydroxyandrostane did not produce an increase in IL-13 as observed in dexamethasone treated animals.

process IL-13 (pg / ml) Saline Control 220 Ovalbumin 230 Vehicle (35 days) 220 Dexamethasone (35 days) 340 3α, 16α, 17α-trihydroxyandrostane (day 35) 195 Vehicle (38 days) 190 Dexamethasone (day 38) 390 3α, 16α, 17α-trihydroxyandrostane (day 38) 210

In addition to decreasing IL-13 rebound at 38 days post-challenge, mice treated with 3α, 16α, 17α-trihydroxyandrostane showed IL-5 levels in lung tissues compared to dexamethasone treated group (145 pg / ml). Reduced (90 pg / ml). IL-5 levels in vehicle control group were 75 pg / ml at 38 days. Other compounds of formula 1 described herein may also be used in this way to identify the ability to treat or alleviate inflammation without IL-13 and / or IL-5 rebound side effects, including 3β, 16β, 17β-trihydroxyandrostan, 3β, 16α, 17α-trihydroxyandrostan, 3β, 16β, 17α-trihydroxyandrostan, androst-5-ene-2α, 3β, 16α, 17β-te Troll androst-5-ene-3β, 7β, 16α, 17β-tetrol and 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol. These results show that the compound of formula 1 can treat lung inflammation or asthma in vivo.

In another protocol, mast cell populations were cultured from murine animal bone marrow as follows. Briefly, bone marrow was extracted from the femur using PBS and 27 g needles from Balb / C mice. The cells were cultured in a mixture of cells secreting 2/3 RPMI-1640 with 19% FBS, and IL-3. Bone marrow cells were allowed to differentiate for 18-25 days in the IL-3 containing mixture prior to use in the experiment. Bone marrow cells cultured in this manner were similar in phenotype to mucosal mast cells and were termed bone marrow cell derived mast cells (BMMC).

Homogeneity of mast cells propagated in vitro was confirmed by analysis with conventional flow cytometry techniques and markers specific for cell type. At 14 and 21 days of proliferation, mature mast cells were harvested and prepared as test cultures. The goal was to determine the effect of compounds such as dehydroepiandrosterone on degranulation coupled to mast cell stimulation. Prepared mast cells were suspended in test culture wells at a density of 1 × 10 ⁷ cells / mL. In control cultures, mast cells induced degranulation after crosslinking of the IgE receptor with the IgE antigen-antibody complex. In the co-culture group, mast cells were dehydroepiandrosterone, which had been pre-cultured at various doses and then activated using anti-IgE antibodies. As a result of measuring the release of β-glucuronidase from the cytoplasmic storage granulocytes of the cells in the absence of stimulation, no degranulation of detectable mast cells was observed. The introduction of anti-Ig-E receptor antibodies to the culture resulted in the release of significant amounts of β-glucuronidase. When mast cells were exposed only to dehydroepiandrosterone alone, no measurable degranulation was observed. However, before activation with the anti-IgE antigen-antibody complex, mast cells that had been previously exposed to 100 μM dehydroepiandrosterone for 5-10 minutes had approximately 70% inhibition of degranulation. Lower levels of dehydroepiandrosterone resulted in a lower proportion of degranulation inhibition. In a similar protocol, 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androst-5-ene-3β, 7β, 16α, 17β-tetrol or androast-5-ene-3α Compounds of Formula 1 such as, 7β, 16α, 17β-tetrol were 10-1000 times more potent than dehydroepiandrosterone.

Example 3

Treatment of fatal infections / shocks. Two compounds, 16α-bromoepiandrosterone (3β-hydroxy-16α-bromoandrostan-17-one) and 3β, 16α-dihydroxy-17-oxoandrostane, were used in the lethal shock protocol. . In one protocol, 3 mg of 16α-bromoepiandrosterone was administered to one group of animals via oral feeding, while another group of 3 mg of 16α-bromoepiandrosterone was administered by subcutaneous injection. It was. Groups of control animals received placebo. In this protocol, 16α-bromoepiandrosterone was administered to mice 24 hours before and 1 hour after the lethal dose of bacterial lipopolysaccharide (LPS). By the end of the observation period, all placebo-controlled animals that received vehicle at 72 hours after LPS administration died, but 65% of the animals that received oral 16α-bromoepiandrosterone survived. Animals that received 16α-bromoepiandrosterone subcutaneously survived 50% of them. Animals that survived for 72 hours recovered from LPS exposure.

In a second experiment, 16a-bromoepiandrosterone or 3β, 16α-dihydroxy-17-oxoandrostane was injected by oral gavage 24 hours before and 1 hour after lethal doses of LPS. Was administered. Vehicle groups treated with vehicle were used as placebo controls. At 72 hours, 25% of the placebo controls survived, 50% of the groups treated with 3β, 16α-dihydroxy-17-oxoandrostane survived and were treated with 16α-bromoepiandrosterone. 80% survived.

In other experiments, the protective ability of 16α-bromoepiandrosterone and 3β, 16α-dihydroxy-17-oxoandrostane against lung wounds caused by exposure to LPS less than lethal doses of mice Tested. In this assay, compounds, sterile saline (negative control) or vehicle (vehicle control) were administered via oral ingestion to the respiratory tract and lungs of animals under mild anesthesia, 24 hours before and 1 hour after administration of 100 μg of LPS. The mice were administered to a group of mice. At 48 hours the animals were sacrificed and samples from the lungs of the animals were obtained from bronchoalveolar alveolar lavage fluid (BAL). The number of cells in the BAL fluid was counted, which means that the lungs were infected and damaged. In this assay, cells that mediate inflammation and lung injury infiltrated the lungs in response to the presence of LPS. For the negative control and vehicle positive control, the BAL fluid contained about 6 × 10 ⁷ cells / mL. The number of cells in the group of animals treated with 16α-bromoepiandrosterone (p = 0.02) or 3β, 16α-dihydroxy-17-oxoandrostane (p = 0.04) significantly affected the number of cells in BAL fluid. Reduced to about 4.4 x 10 ⁷ cells / mL. These results indicate that the compound is active in clinical symptoms such as asthma or COPD in which lung wounds or damage are uncontrolled or associated with excessive infection. Other compounds, such as 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol or 17β-aminoandrost-5-ene-3β-ol, can also be used to characterize in a similar manner.

Example 4

Clearance of bacteria from lung tissue. The ability of 16α-bromoepiandrosterone to eliminate Pseudomonas aeruginosa infection in lung tissue has already been disclosed, namely A. van Heeckeren et al., J. Clin. Invest ., 100 (11): 2810-2815 1977; A. van Heeckeren et al., Am. J. Respir. Crit.Care Med ., 161: 271-279 2000). This protocol was performed in CFTR mice, which was used as an animal model for human cystic fibrosis (SD Freedman et al., Proc. Natl. Acad. Sci. USA , 96 (24): 13995-14000 1999; W Zeng et al., Am. J. Physiol.Cell . Physiol . 273: C442-C455 1997). The establishment of chronic Pseudomonas aeruginosa infections using agarose beads containing bacteria (50 μl containing about 6.1 × 10 ⁴ CFU / animal) is already disclosed (JR Starke et al. , Pediatr Res, 22:.. 698- 702 1987). Two groups of mice (n = 9 for each group) were treated with 40 mg / kg of 16α-bromoepiandrosterone or vehicle (control) and the lungs of animals 10 days after the introduction of agarose beads into the lungs The bacterial burden of was measured. On day 10, the bacterial burden in the lungs of the vehicle control animals was about 6 × 10 ⁶ CFU / animal, while animals treated with 16α-bromoepiandrosterone had reduced bacterial burden (p = <0.04). These results indicate that 16α-bromoepiandrosterone may be used to treat or alleviate pulmonary infections, as well as anti-inflammatory effects, which may be used for the treatment of inflammation and cystic fibrosis, in which the infection may be present at the same time. Which is a desirable therapeutic agent.

Example 5

Anti-inflammatory activity of human cells in vitro. The ability of 16α-bromoepiandrosterone and 3β, 16α-dihydroxy-17-oxoandrostane to reduce inflammation in human cells in vitro was measured using human whole blood exposed to LPS. Without compounds, 16α-bromoepiandrosterone (100 ng / mL) and 3β, 16α-dihydroxy-17-oxo, compared to cells exposed to LPS alone (positive control) or vehicle (dimethylsulfoxide) Reduced production of γ-interferon by cells was observed in the presence of androstane. The amount of γ-interferon in the growth medium was measured when the cells were incubated for 24 hours in the presence of LPS.

Example 6

Treatment of autoimmune neurodegeneration. Three compounds, namely 17β-aminoandrost-5-ene-3β-ol, 17β-dimethylaminoandrost-5-ene-3β-ol and 17β-methylaminoandrost-5-ene-3β-ol, Mice were characterized for their ability to alleviate experimental allergic encephalomyelitis (EAE). These demyelinating conditions are commonly used as models for multiple sclerosis in humans and are used to test new therapeutic agents for the treatment of multiple sclerosis (eg, BF Bebo Jr. et al., J. Neurosci Res . 52: 420-26 1998; RR Voskuhl et al., Neuroscientist, 7: 258, 270 2001; H. Offner et al., J. Neuroimmunol ., 130: 128-139 2002). Activation in this model indicates that the test compound is capable of slowing the rate of neuronal death associated with the progression of EAE disease, or preventing neuronal death.

In this protocol, compounds were administered to female SJL / J mice by oral feeding at the onset of disease symptoms. Antigen was used to initiate EAE symptoms in mice. The antigen used for active immunization was mouse proteolipide protein (PLP) 139-151 (HCLGKWLGHPDKF). Immunization with this peptide antigen initiates autoimmune Th1-mediated demyelinating diseases of the central nervous system. Antigens were prepared by solid phase synthesis and purified by high performance liquid chromatography. EAE symptoms began in female SJL / J mice by immunizing with 150 μg of PLP 139-151 peptide in a complete Freund adjuvant containing 200 μg of Mycobacterium tuberculosis . Immunization protocols were performed on day 0 by subcutaneous injection on 4 sites on the posterior flank. After immunization, the following scores were used to assess daily clinical signs of EAE: 0 = no clinical signs, 1 = sag tails; 2 = weakly hind limb weakness and limp tail; 3 = medial hind limb weakness and sagging tail or weak ataxia, 4 = severe hind limb weakness and mild forelimb weakness and moderate ataxia, 5 = moderate forelimb weakness or paralysis; 6 = Paralysis with severe forefoot weakness or severe ataxia or wandering.

Mice in the vehicle control group started showing observable symptoms of EAE about 10-11 days after immunization with PLP antigens (which is typical for EAE disease models). Daily 17β-aminoandrost-5-ene-3β-ol, 17β-dimethylaminoandrost-5-ene-3β-ol or 17β-methylaminoandrost-5-ene-3β- starting at day 1 post immunization Ol was administered via oral feeding. All three compounds were active at the dose of 5 mg / kg and had a lower clinical severity of symptoms observed at day 26 at the end of the observation period. Therapeutic activity of this compound was observed at a blood concentration of about 10 μg / ml in mice. These results indicate that the compound was biologically active in treating autoimmune neurodegenerative diseases.

Example 7

Inhibition of NF-κB in vitro. A number of compounds have been used to inhibit the activation of NF-κB by TNF-a or LPS in human cells in vitro. Activation of NF-κB increases the expression of many genes that mediate inflammation. This protocol used human THP-1 cells, which are human mononuclear blood cells of the monocyte phenotype. A cell line called NF-κB-bla THP-1 contains a β-lactamase reporter gene under the control of the NF-κB response element (Invitrogen, CellSensor ^™ , product No. K1176). In this cell line, the β-lactamase reporter gene is stably integrated into THP-1 cells. This cell line was used to detect agonists or antagonists of the NF-κB signaling pathway. These NF-κB-bla THP-1 cells responded to the presence of tumor necrosis factor α (TNFα) or bacterial lipopolysaccharide (LPS) by increasing the expression of β-lactamase reporter gene. The level of β-lactamase enzyme activity was measured by fluorescence resonance energy transfer ratiometric detection. TNFα and LPS are efficacious as inflammatory agents that activate NF-κB in THP-1 cells. In this assay, a compound that reduces NF-κB activity, and hence β-lactamase activity in the presence of TNFα or LPS, possesses anti-inflammatory activity.

NF-κB-bla THP-1 cells were maintained by passaging as needed. Cells grown in suspension were maintained at a density of 2 × 10 ⁵ cells / ml to 2 × 10 ⁶ cells / mL. Cells were placed in 384-well black-wall, clear bottom assay plates (Costar # 3712-TC low fluorescence background plates) at 20,000 cells / well 24 hours prior to adding 10 ng / mL of TNFα or 0.2 ng / mL of LPS. Inoculation was performed to activate NF-κB. In positive control assays to activate NF-κB, the EC ₅₀ concentration for TNFα was 0.20 ng / mL 1 hour after β-lactamase substrate culture. The EC ₅₀ dose for LPS was 0.15 ng / mL. The EC ₅₀ concentration for TNF-α or LPS is defined as 50% of the TNF-α or LPS concentration causing the maximal activation of NF-κB in this test. Synthetic glucocorticoid dexamethasone (a potential anti-inflammatory drug) reduced the effect of TNFα with an EC ₅₀ of 0.47 nM (average of five assays) in this assay. Similar biological activities for dexamethasone have been reported in other in vitro cell assays, which completely inhibited NF-κB activation at IC ₅₀ of about 1 nM (MKA Bauer et al., Eur. J. Biochem . 243: 726- 731, 1977).

Using this assay, the IC ₅₀ of the compound for inhibition of NF-κB activation in NF-κB-bla THP-1 cells after LPS stimulation is shown below. IC ₅₀ concentration of a compound used in this assay means a concentration of a compound that causes 50% of the maximum inhibition of NF-κB activity that the compound can induce. This assay is usually performed 2 to 4 times for each compound, the values of which are shown below as averages for each compound. The data in Table 1 show that with very low levels of many such compounds, it is possible to inhibit NF-κB in human macrophages.

IC ₅₀ ^* compound 0.47 nM ± 0.11 Dexamethasone (positive anti-inflammatory control) > 10 μM Estradiol (negative anti-inflammatory control) 8.2 fM ± 7.4 3β, 7β, 16α, 17β-tetrahydroxyandrost-5-ene 84.5 fM ± 65 3α, 7β, 16α, 17β-tetrahydroxyandrost-5-ene > 10 μM 3β, 7α, 16α, 17β-tetrahydroxyandrost-5-ene > 10 μM 16α-acetoxy-3β, 7β, 17β-trihydroxyandrost-5-ene 0.4 fM 3β, 4β, 16α, 17β-tetrahydroxyandrost-5-ene 0.01 fM 4β-acetoxy-3β, 16α, 17β-trihydroxyandrost-5-ene 2.0 fM 3β-acetoxy-7β, 11β, 17β-trihydroxyandrost-5-ene > 10 μM 3β, 7β, 11β, 17β-tetrahydroxyandrost-5-ene 10 fM 3β, 7β, 17β-trihydroxy-11-oxoandrost-5-ene 0.1 pM 17α-methyl-3β, 11α, 17β-trihydroxyandrost-5-ene > 10 μM 3β, 11α-dihydroxy-17-oxoandrost-5-ene 2.0 fM 2α, 3β, 17β-trihydroxyandrostan 14 pM ± 12 3β, 17β-dihydroxyandrost-5-ene 1.2 fM ± 0.28 3β, 7β, 17β-trihydroxyandrost-5-ene > 10 μM 3β, 7α, 17β-trihydroxyandrost-5-ene 19 fM ± 11 3β, 7β, 17β-trihydroxy-17α-ethynylandrost-5-ene > 10 μM 3β, 7β, 17β-trihydroxy-17α-trifluoromethylandrost-5-ene 6.8 fM ± 5.6 3β, 7α, 17β-trihydroxy-17α-ethynylandrost-5-ene 12 fM ± 9.8 3β, 7β, 17β-trihydroxy-17α-vinylandrost-5-ene 50.3 fM ± 13.9 3β, 7β, 17β-trihydroxy-17α-methylandrost-5-ene 64 fM ± 36 3β, 7α, 17β-trihydroxy-17α-methylandrost-5-ene 30 pM ± 29 16α-fluoroandrost-5-ene-17-one 1.9 nM ± 0.8 16α-idodoepandrosterone 8.8 μM ± 1.3 16α-bromoepiandrosterone 0.6 μM ± 0.2 16β-bromoepiandrosterone > 10 μM 16α-hydroxyepiandrosterone 7.2 fM ± 4.7 3β, 17β-dihydroxy-17α-methylandrost-5-ene 11.5 fM ± 3.5 3β, 17β-dihydroxy-7-oxo-17α-ethynylandrost-5-ene > 10 μM 3β, 17β-dihydroxy-7-oxo-17α-methylandrost-5-ene * μM = 10 ^-6 M; nM = 10 ^-9 M; pM = 10 ^-12 M; fM = 10 ^-15 M

Other compounds showing anti-inflammatory activity in this protocol are 3α-pentafluoroethylandrost-4-ene-3β, 17β-diol (IC ₅₀ 3.1 nM), 3α-pentafluoroethylandrost-5-ene-3β , 17β-diol (IC ₅₀ 17 nM; maximum NF-κB inhibition was 50%), 3α / β, 17α-ethynylandrostan-3α / β, 17β-diol (IC ₅₀ 200 pM), 17α-trifluor Chloromethylandrostan-3α, 17β-diol (IC ₅₀ 190 nM), 17β-glyciandroandrostan-3β-ol (IC ₅₀ 0.42 pM), 3β-glycyandrostan-17β-ol (IC ₅₀ 1 nM) , Androstane-3β, 16β-diol-17-oxime (IC ₅₀ 1.9 fM) 17α-ethynylandrost-4-en-3-one-17β-ol (IC ₅₀ 2.9 fM; maximum NF-κB inhibition was 80 %), 16α-fluoroandrost-5-en-17-one (IC ₅₀ 30 pM), 16β-fluoroandrost-5-en-7β-ol-17-one (IC ₅₀ 1.5 nM), Androstane-3α, 16α, 17β-triol (IC ₅₀ 6.9 fM), Androstane-3α, 16β, 17β-triol (IC ₅₀ 19 fM), Androst-5-ene-3β-ol-17β-succinct Nyl Ester (IC ₅₀ 0 .2 nM), 3β-acetoxy-7β, 17β-dihydroxy-11-oxoandrost-5-ene (IC ₅₀ 1 fM; maximum NF-κB inhibition was 65%). The maximum inhibition of NF-κB by this compound was about 25% to 80%, which is different from the 100% inhibition of NF-κB by synthetic glucocorticoid dexamethasone in this protocol.

Two compounds with increased NF-κB activity in this protocol were androst-5-en-3β, 7α, 16α-triol-17-one (IC ₅₀ 1.3 nM; 140% NF-κB activity compared to control cells). And 3β, 17α-dimethylandrostan-3α, 17β-diol (IC ₅₀ 40 nM).

Compounds that do not exhibit anti-inflammatory activity in this protocol include 3α, 17α-methylandrostan-3β, 17β-diol, 3β-acetoxyandrost-5-ene-3β, 17β-diol, 17α-methylandrost-5- En-3β, 17β-diol-7-one, 16α-fluoroandrost-5-ene-7β-ol-17-one, 16α-fluoroandrost-5-en-7α-ol-17-one, 17α-methylandrostan-3β, 7α, 17β-triol, androast-5-ene-3β, 11β, 17β-triol, 16α-fluoroandrostan-17-one, androast-5-ene-3α , 17β-diol, androstane-2β, 3α, 16α, 17β-tetrol and androstan-3α, 16α, 17α-triol, all of which were IC ₅₀ > 10 μM.

The ability of compounds to reduce the activity of NF-κB in small amounts is an important factor in the treatment of anti-inflammatory, especially in diseases where excessive levels or nuclear transcriptional activity mediated by NF-κB are important in the pathogenesis of disease or condition. Implying that it can be used.

In the assay method described above, the maximum inhibition rate of NF-κB by dexamethasone, 16α-bromoepiandrosterone and 16β-bromoepiandrosterone was 100% and NF-κB detectable at concentrations of IC ₅₀ for these compounds. No activity was observed. In contrast, other compounds such as 3β, 7β, 16α, 17β-tetrahydroxyandrost-5-ene, 3α, 7β, 16α, 17β-tetrahydroxyandrost-5-ene or 3β, 7β, 17β-tri The maximum inhibition rate of NF-κB by hydroxy-17α-methylandrost-5-ene was less than about 80%, which was significant additional to NF-κB activity, even though the amount of compound increased above the IC ₅₀ value. Did not provide an effect. For most of these compounds, the maximum degree of inhibition of NF-κB in this assay was about 25 to 80%, mainly about 30 to 65% or about 30 to 70%. These results indicate that compounds such as 3β, 7β, 16α, 17β-tetrahydroxyandrost-5-ene inhibited NF-κB activity through a mechanism that could not completely inhibit its biological activity.

Some of the compounds in Table 1 did not have detectable ability to possess anti-inflammatory activity in in vitro cell assays. Other compounds tested but not active in this assay (IC ₅₀ > 10 μM) include 3β, 17α-dihydroxyandrost-5-ene, dehydroepiandrosterone (3β-hydroxyandrost-5-ene- 17-one), 3β-hydroxyandrostan-7,17-dione, 16α-bromo-3β, 17β-dihydroxyandrost-5-ene and 16β-bromo-3β-hydroxyandrost-5 There is a yen-17-on. Nevertheless, some of the compounds that were inactive in this in vitro cell assay, such as 16α-hydroxyepiandrosterone, have been found to have anti-inflammatory activity in animals in vivo. These results show that these compounds can be activated through other mechanisms or require cellular abnormalities from a single cell line. In vivo anti-inflammatory activity of such compounds can be derived, for example, by inducing prostaglandin synthesis and other activities in the liver, leading to systemic anti-inflammatory responses. Alternatively, the anti-inflammatory activity for these compounds may be derived from the ability of the compounds to inhibit stimulation of NF-κB activity, which may be derived from sources other than LPS. A number of different substances can activate the activity of NF-κB, including the presence of LPS, TNF-α, IL-1, viral or bacterial gene products, activation of B- or T-cells, the activation of cells in ultraviolet light. Exposing and the like. Not all cell types respond to all of these stimuli because not all cells express the signaling mechanisms required to respond to each stimulus. Most types of cells can respond to some of these signals, but in rare cases a given cell may respond to all stimuli. In the assays used herein, activation of NF-κB results from stimulation induced by bacterial LPS, such that compounds with limited ability to inhibit LPS signal transduction pathways inhibit NF-κB activity in this assay. It is expected to have a limited ability to reduce.

Methods of modulating NF-κB that may be used or included in the practice of the present invention include those described in the following publications. U.S. Pat.Nos. 5,989,835, 6,410,516, 6,545,027, 6,831,065 and 6,998,383. Other aspects of NF-κB activity have also been described and can be included in the methods of the invention (eg, AS Baldwin, Annual Rev. Immunol . 14: 649-683 1996; M. Muller et al., Mol. Cell. Biol 22 ((4) 1060-1072 2002; PA Baeuerle, Cell 95: 729-731, 1998).

Example 8

The ability of selected compounds to treat LPS induced shock / inflammation in mice was examined using protocols similar to those described above. Five groups of three ICR mice weighing approximately 30 g each were treated with 120 μl vehicle (30% sulfobutylether-cyclodextrin in water) and androst-5-ene-3α, 7β, 16α, 17β-te in vehicle By intraperitoneal injection of troll, androast-5-ene-3β, 4β, 16α, 17β-tetrol in vehicle, or 4β-acetoxyandrost-5-ene-3β, 16α, 17β-triol, respectively, in vehicle Treated. All drug and vehicle formulations were solutions and not in suspension. Sulfobutylether-cyclodextrin is commercially available (Captisol ^™ , www.cydexinc.com). There were two vehicle control groups, one with vehicle only and the other with vehicle and LPS. Vehicle and drug were administered 24 hours before and 1 hour after LPS (approximately LD _50/24 administration, ie 50% lethal dose 24 hours after LPS administration) by intraperitoneal injection. The drug was administered at about 40 mg / kg (1.2 mg drug / animal for each dose of drug). 1.5 hours after LPS injection, the spleen was taken out of the animal to degrade the spleen cells, the nuclei were separated from the spleen cells, and NF-κB was measured from the digested nuclei, and analyzed for activated NF-κB. As a result, in all three compounds, NF-κB activity was reduced by about 50% compared to LPS + vehicle control. The level of activated NF-κB in spleen cells in animals treated only with vehicle and not with LPS was essentially the same as the activated NF-κB in spleen cells from drug-treated animals. This result means a potential anti-inflammatory effect in animals as evidenced by the reduction of activated NF-κB in drug treated animals compared to control animals.

Other compounds used in the same protocol that analyzed NF-κB or TNFα after 1.5 hours of LPS challenge include androstan-3α, 16α, 17β-triol, androstan-3α, 17β-diol-16-one, 17α-tri Fluoromethylandrost-5-ene-3β, 17β-diol, androast-5-ene-3α, 17β-diol, androst-5-ene-3α, 16α, 17β-triol, 3α-trifluoro Methylandrost-5-ene-3β, 17β-diol, androstan-3α, 17β-diol-16-oxime and androast-5-ene-3β, 17β-diol-7-one. All of these compounds were administered as solutions (not suspensions) of the compound with sulfobutylether-cyclodextrin in water. These compounds were administered 24 hours before the LPS challenge and concurrently with the LPS challenge (instead of 1 hour after the LPS challenge as described above), followed by analysis of NF-κB and blood TNFα in the spleen with LPS Challenge 1.5 It was performed after hours. Compounds were administered at 40 mg / kg and 4 mg / kg, 0.1 mg / kg and 0.05 mg / kg for 3α-trifluoromethylandrost-5-ene-3β, 17β-diol. Inhibition of NF-κB and TNFα was observed for both of these compounds compared to the vehicle control. For 3α-trifluoromethylandrost-5-ene-3β, 17β-diol, maximum inhibition of NF-κB and TNFα was observed in animals treated at the 0.1 mg / kg dose.

Example 9

Epidemiologic analysis of NF-κB inhibition in vivo. The mechanism of action of compounds such as 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol was further examined by examining the dynamics of NF-κB inhibition after injection of bacterial LPS into mice. The compound will at least partially inhibit the activation of NF-κB induced by LPS or TNFα in immune cells (macrophages or monocytes) in vitro as described in Example 7. In this study, mice received 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol (about 40 mg / kg, about 1.2 mg / animal) of the compounds in the vehicle described in Example 8. The solution (not suspension) was injected intraperitoneally. Drugs were injected 24 hours prior to intraperitoneal injection of bacterial LPS (about LD _50/24 ). The study used two groups of twelve animals, each of which was given the drug 24 hours prior to vehicle control or LPS challenge. Immediately before the LPS challenge and after 1.5, 2.0 and 2.5 hours after LPS administration, spleens were removed from three animals from both groups. Splenocytes were obtained and the levels of activated NF-κB measured essentially by the assay of NF-κB in the nucleus, essentially as described in Example 8.

Maximal NF-κB inhibition after LPS administration occurred at 1.5 hours in the vehicle control, which was a 4-fold increase over the pre-LPS level of activated NF-κB. The results are shown below. Values for vehicle control and drug treated animals are expressed in relative optical density units from values measured by ELISA of NF-κB in the nucleus in spleen cells.

Time (hours) Vehicle control Drug treatment group 0 18 22 1.5 72 2 2.0 10 7 2.5 10 9

Significant inhibition of NF-κB at the 1.5 hour time point and relatively normal levels of NF-κB activity at other time points have the compound having activity that is transient but potentially capable of inhibiting LPS-induced trauma in a critical period after LPS exposure. It is meant to mean. Similar assays in other studies showed that levels of activated NF-κB at 30 and 60 minutes after LPS injection in vehicle control mice were similar to the pre-LPS time point in this study. These results indicate that in this model, the effect of LPS on the activation of NF-κB in splenocytes is about 1.5 hours after LPS challenge. This time point represents a convenient time or window, ie the time at which the activity of the anti-inflammatory drug candidate can be measured in vivo, ie about 75 minutes to about 105 minutes after the LPS challenge. The window may vary depending on the route of administration of the biological injury, eg, the route of administration of the biological injury, such as LPS or TNFα administered by intraperitoneal injection, as compared to LPS or TNFα administered by subcutaneous or intramuscular injection. Similarly, a biological injury may result in the subject being exposed to ionizing radiation at about 50-60 cGy / min, for example about exposing about 500-1000 cGy / min or about 5-10 cGy / min of ionizing radiation to the subject. Other factors, such as exposing.

Analysis of TNFα expression induced by LPS in mice showed the highest TNFα level after 1.5 hours of LPS challenge (administering 500 μg of LPS by intraperitoneal injection) among the TNF alpha levels observed 1-2 hours after LPS challenge. Is showing. The RNFα level was highest 30 minutes after LPS administration and decreased after 2.5 hours.

Other compounds that can be analyzed for their ability to act as biomechanical drugs include androast-5-ene-3β, 7β, 16α, 17β-tetrol, androast-5-ene-3α, 7β, 16α, 17β- Tetrol, androst-5-ene-3β, 7α, 16α, 17β-tetrol, androast-5-ene-3β, 4β, 16α, 17β-tetrol, androast-5-ene-3β, 4α, 16α, 17β-tetrol, androst-5-ene-3α, 4β, 16α, 17β-tetrol, 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, 17α-ethynylandro Strut-5-en-3β, 7α, 17β-triol, 17α-ethynylandrost-5-ene-3β, 17β-triol-7-one and pharmaceutically acceptable analogs of any of these compounds Such as, for example, analogs that are hydrosyl esters or ether derivatives in one, two, or more hydroxyl groups. Suitable esters and ethers include acetate, n -propionate, i -propionate, succinate, -OC (O)-(CH ₂ ) _n -CH ₂ R, -OC (O) -O- (CH ₂ ) amino acids such as _n- CH ₂ R, -OC (O) -NH- (CH ₂ ) _n -CH ₂ R, glycine and alanine (-OC (O) -CHCH ₃ -COOH), hydroxy esters and methyl, ethyl , n -propyl, i -propyl -O- (CH ₂ ) _n -CH ₂ R, -O- (CH ₂ ) _n -O-CH ₂ R (e.g. -O-CH ₂ CH ₂ -O-CH ₃ ) Ether, where n is 1, 2, 3, 4, 5 or 6, and R is -H, -F, -Cl, -Br, -I, -OH, -C (O) OH (or Acceptable salts thereof, such as sodium or potassium salts), -C (O) OCH ₃ , -C (O) OC ₂ H ₅ .

Example 10

The ability of compounds of Formula 1 to influence the progression of arthritis in a passive collagen-induced arthritis model was examined in essentially the same manner as previously described (E. Simelyte et al., Arthritis & Rheumatism). , 52 (6): 1876-1884, 2005; Z. Han et al., Arthritis & Rheumatism 46 (3): 818-823, 2002; H. Miyahara et al., Clin. Immunol.Immunopathol ., 69 (1 ): 69-76, 1993). In this protocol, passive collagen induced arthritis was induced in DBA / 1 mice by administering an anti-type II collagen antibody, which induces an immune response against joint tissue in the animal. Efficacy in this arthritis model is mainly shown for inflammation, which is measured separately from the intracellular effects operable in arthritis. The severity of arthritis was measured using a semiquantitative clinical scoring system. In groups consisting of 8 animals per group, 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol was treated at 40 mg / kg / day for 14 days or via oral feeding. The vehicle was treated for 14 days. Vehicle was 30% cyclodextrin-sulfobutylether in water and drug solution was vehicle solution containing 20 mg / ml drug.

Animals were examined by measuring ankle thickness and arthritis score (4 points / paw), with higher scores indicating more severe arthritis. The experiment was finished after about 14 days and histology and gene expression measurements were performed. For histological examination, the left hind paw was obtained, fixed in 10% formalin for 24 hours, decalcified and then embedded in paraffin. Tissue sections were stained with hematoxylin and eosin for safranin O-fast green to determine proteoglycan content. A semi-quantitative scoring system was used to measure inflammation, extra-articular inflammation, erosion and proteoglycan loss of joint synovial fluid.

Treatment with the compound was performed after administration of the antibody. By the protocol, the effect of treatment on the progression of arthritis could be observed. As a result, collagen-induced arthritis in group 1 was reduced in group 1 animals compared to group 4 animals, even on days 7-14. The maximum clinical score in vehicle treated animals was 10.2 points on day 8, compared to 5.1 on the maximum clinical score on day 7 in group 1. On the 14th day of the last day of the protocol, the clinical score of the vehicle treated group was 7.8 and the control score was 4.1. Differences in clinical scores between 7 and 14 days were apparent in the treated animals, indicating that there was a reduced level of inflammation in the treated animals compared to the vehicle control animal group. The effect of treatment with 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol was similar to that treated with dexamethasone, which alleviated the severity of arthritis and inhibited inflammation in this animal model. The ability to alleviate the severity of arthritis of 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol has been shown to reduce the efficacy of cell mediated immunity such as methotrexate or anti-TNFalpha therapeutics, which have low efficacy in this arthritis model. Contrast with suppressor.

Example 11

The ability of the compound of formula 1 to investigate LPS-induced lung wounds in mice was investigated. LPS-induced lung wound models have already been used to evaluate treatments for acute lung wounds (ALI), acute adult respiratory distress syndrome (ARDS) and endotoxin shock or sepsis (Metz et al., C., Chest 100 (4): 1110-9, 1991; Windsor, AC et al., Ann. J. Med. Sci. 306 (2): 111-6, 1993; Brigham KL et al., Am. Rev. Respir. Dis 133 (5): 913-27, 1986).

This protocol was performed substantially the same as described in Su, X. et al., Intenstive Care Med. 30: 133-140, 2004. Female 6-8 week old C57 / BL6 mice (average weight 25 g) were purchased from Jackson Labs (Bar Harbor, ME) and randomly divided into groups of 7 animals and maintained under standard food and cage conditions. . The groups were divided into: (1) saline and LPS treated mice, (2) vehicle and LPS treated mice, (3) 125 μg dexamethasone treated mice, (4) 40 mg / Kg androst- Mice treated with 5-ene-3β, 7β, 16α, 17β-tetrol and LPS, (5) mice treated with 40 mg / Kg 5α-androstan-3β, 16α-diol-17-one and LPS, ( 6) mice treated with 40 mg / Kg 5α-androstan-3β, 17β-dihydroxy-16-oxime, (7) 40 mg / Kg androast-5-ene-3α, 7β, 16α, 17β-tet Rolled mouse.

On day 1 mice were pretreated with the compound or vehicle. On day 0, mice were treated with a second amount of compound or vehicle. At 0 + 60 days, 100 μg of E. coli, with a light anesthesia, illuminating the trachea straight. E. Coli LPS (Sigma) was challenged. On day 2 (ie 48 hours after LPS challenge), mice were sacrificed and BALs were obtained (where cell numbers and TNFα / IL6 levels were measured). Lungs were removed, chopped and used for myeloperoxidase (MPO) studies. LPS-induced acute lung infection procedure by dropping 50 mg LPS ( E. Coli 0111: B4, Sigma-Aldrich) in 100 ml PBS dropwise to trachea of weakly anesthetized (isoflurane) mice Was performed. Mice were sacrificed after 48 hours. After that, the bronchial incision was performed after exposing the bronchus under the neck. A blunt 20 gauge needle was inserted into the exposed bronchus and tied to it to obtain bronchial lavage fluid (BAL). In order to minimize bleeding and trauma in the airways, BAL was performed using 0.5 ml of sterile PBS three times. A total of 1300 ml is usually obtained in this procedure. Differentiated leukocyte counts of cells were measured in BAL fluid (BALF) using a hemocytometer. 80-100 cells were differentiated. After obtaining BAL, the thoracic cavity was opened and 3 ml saline was injected into the heart / lung using R ventricular puncture. All lung tissues were harvested and prepared for MPO analysis. For this assay, lungs were homogenized in potassium phosphate buffer (containing 0.5% hexadecyltrimethylammonium bromide pH 6.0). After centrifugation (14,000 × g, 10 min, 4 ° C.), 50 μl of the supernatant was added with 950 μl of potassium phosphate buffer containing 0.2 mg / ml o-Dianisidine dihydrochloride (Sigma-Aldrich) and 0.00002% hydrogen peroxide. Added. Absorbance change was observed at 460 nm. Cytokine levels were measured in BALF cell-free supernatants (200 × g, 10 min, 4 ° C.) by performing ELISA against TNFα, IL-6 (R & D Systems) using a commercial ELISA. Of particular note is the results for androst-5-ene-3β, 7β, 16α, 17β-tetrol, which indicate that in animals treated orally with this compound, MPO, TNFα in BAL compared to animals treated with vehicle. And IL-6 levels were reduced. The effect on MPO, that is, the effect on the measurement of neutrophils in the lung and the pro-inflammatory cytokine TNFα, was particularly good. These results support the compound's ability to reduce proinflammatory cytokine signaling, as well as the ability of the compound to block proinflammatory cells from traveling to infected tissues. In this model, acute inflammation is likely to arise from the innate immune component being stimulated by LPS. Many of the same mediators are believed to be increased and involved in pulmonary inflammation associated with severe disease, which includes certain infectious diseases such as cystic fibrosis, chronic obstructive pulmonary disease, acute and chronic bronchitis, and tuberculosis. Treatment with androst-5-ene-3β, 7β, 16α, 17β-tetrol significantly reduced MPO and pro-inflammatory cytokine levels at 48 hours in BALF, a specific disease model of chronic inflammation, including EAE. Androast-5-ene-3β, 7β, 16α, 17β-tetrol refers to maintaining the proinflammatory effects reported in the present invention.

Example 12

Human mixed lymphocyte reaction (MLR). 3β, 16α-dihydroxy-17-oxoandrostane, 3β, 17β-dihydroxy-16-oxoandrostane, 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol and 17β- Aminoandrost-5-ene-3β-ol was tested for its ability to antigen-specific stimulation in human T lymphocytes in response to specific foreign antigens (major histocompatibility complex). MLR is used as an in vitro model of delayed hypersensitivity reactions and shows possible effects on human antigen specific T cell responses in vivo. Inhibition of MLR by the compound shows the immune suppressive response of the compound to lymphocytes. Compounds that do not inhibit MLR are not immunosuppressive to antigen specific activity in response to lymphocytes.

Blood samples were obtained from fasting, healthy 23-31 year old human volunteers of three (two males, one female). Subjects did not take immunomodulators, anti-allergic or antibiotics for 3 months prior to the start of the study. Blood was drawn from the subject between 9 and 10 am to limit possible changes in blood levels of hormones or cytokines that could affect the in vitro response to lymphocytes. Capillary mononuclear cells (PBMC) were isolated by centrifugation in Ficoll-Hypaque (density 1.077, Biochrom AG, Berlin, Germany) gradient, culture medium (2 mM L-glutamine, penicillin (100 U / mL) and streptomycin ( Resuspended in RPMI 1640) (Invitrogen s.rl., Milan, Italy) supplemented with 100 mg / mL). Autologous tissue inactivated plasma was used at 10%. Irradiated (30 Gy) irritant PBMCs (PBMCs) of 500 responder PBMCs (PBMCr) and 500,000 allogeneic were mixed in a 1: 1 ratio in 200 μl medium to provide a flat bottom 96 well plate (Nunc, Roskilde). , Denmark) was incubated for 6 hours at a concentration of 300 nM or 30 nM for each of the four compounds. The compound was dissolved in ethanol and then diluted to the desired concentration using the culture medium to make a final solution containing 0.01% ethanol. This vehicle was used as a control. Controls included separately cultured PBMCr and PBMCs. PBMCs were pulsed with 1 μCi / well [ ³ H] thymidine (Amersham, Milan, Italy) during the last 8 hours of incubation period. Cells were then harvested and measured for incorporation of radiation using a beta cell counter. The average cpm of the four measurements was calculated. T cell proliferation was expressed as stimulator: SI = cpm (PMBCs xPBMCr) / cpm (PBMCr) + cpm (PBMCs). Statistical analysis was performed using the Student's t test. The cpm obtained from the four measurements of each test compound was compared with the proliferative response obtained from control PBMCr and PBMCs cultured in the presence of vehicle. A difference of p <0.05 was considered significant.

The test results showed that none of the four compounds inhibited MLR, except for 17β-aminoandrost-5-ene-3β-ol at 300 nM. This is equivalent to 3β, 16α-dihydroxy-17-oxoandrostane, 3β, 17β-dihydroxy-16-oxoandrostane and 17α-ethynylandro-5-ene-3β, 7β, 17β-triol. NM or 30 nM did not inhibit immunity in this assay (p> 0.05), but at 300 nM it indicates that 17β-aminoandrost-5-en-3β-ol inhibited immunity moderately compared to the control response (p <0.05). These results show that 3β, 16α-dihydroxy-17-oxoandrostane, 3β, 17β-dihydroxy-16-oxoandrostane and 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol This indicates that it is not an immune inhibitor against human lymphocytes in vivo. These results are consistent with the compound's ability as an anti-inflammatory agent (eg, Example 7) that does not suppress immunity.

Example 13

Analysis of Immune Suppression. Glucocorticoid steroids such as dexamethasone or hydrocortisone usually inhibit immunity and carry significant toxicity when they are used. Immunosuppression has already been described in C. Goebel et al., Inflamm. Res ., 45 (Suppl. 2): S85-S90, 1996; R. Pieters et al., Environmental Health Perspectives 107 (Suppl. 5): 673- 677, 1999) was examined in reporter antigen popliteal lymph nodes in essentially the same manner as described. This protocol demonstrates that this compound does not have some degree of immunosuppressive activity in vivo, in the popliteal lymph node (PLN) assay of 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol. It was used to analyze the activity. In this protocol, the vehicle contained 0.1% carboxymethylcellulose, 0.9% saline, 2% tween 80, and 0.05% phenol, which was 17α-ethynylandrost-5-ene in suspension in drug-treated animals. -3β, 7β, 17β-triol. Determination of activity includes the following steps: (1) measuring the inhibition in popliteal lymph node cells in the total lymphocyte count, antigen specific IgM, IgG1 and IgG2a antibody secreting cell (ASC) (ELISPOT assay) number, ( 2) Analyzing expression of cell surface markers (CD4, CD8, CD19, F480, CD80, CD86) by flow cytometry of live cells in suspension, (3) IL-4, TNFα and IFNγ by lymphocytes in vitro Measuring production (ELISA).

A group of specific pathogen-free BALB / C mice (n = 5 per group) was used. Positive controls were induced by vehicle (oral feeding) and 5 μg / 1 day dexamethasone by subcutaneous injection to induce an immunosuppressive response. Vehicle control animals (negative controls) were treated with vehicle only (oral feeding). One group of animals was treated with 17α-ethynylandro-5-ene-3β, 7β, 17β-triol at 0.1 mg / day via oral feeding. In another group, 1 mg / 1 day of 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol was administered via oral feeding of animals. The results were analyzed by t-test (two-tailed Student's t-test) using the same variables. 50 μl of a sensitized amount of TNP-OVA prepared immediately was injected into the right rear paw of the animal. Immediately after sensitization with TNP-OVA, dexamethasone (Decadlon phosphate injection; dexamethasone sodium phosphate) was injected subcutaneously in the nape of the neck. 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol was injected immediately via oral feeding. Five days after the injection of TNP-OVA, the eye was punctured to collect blood, the neck of the mouse was torn down, the popliteal lymph nodes were removed and then isolated from the surrounding adipose tissue. Single cell suspensions were prepared, resuspended in 1 ml PBS-BSA (1%) and counted. Cell number, IL-4, IL-5 and IFNγ were measured.

The average number of lymphocytes in PLN from the vehicle control group was 7.8 × 10 ⁶ per lymph node compared to 2.9 × 10 ⁶ per lymph node in the animal group treated with dexamethasone. This reduced lymphocyte count clearly shows a distinct immunosuppressive response as is usually observed with dexamethasone or other glucocorticoid compounds. In contrast, the group treated with 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol at 1 mg / 1 day had 8.2 × 10 ⁶ lymphocytes per lymph node and 0.1 mg / 1 day. The group treated with 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol had 11.1 × 10 ⁶ lymphocytes per lymph node. Tests have shown that 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol does not inhibit immunity but enhances immunity. Treatment with 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol at 1.0 mg / 1 day and 0.1 mg / 1 day increased IFNγ, IL-4 and IL-5 levels compared to vehicle control. To improve immunity. The effect of 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol at 0.1 mg / 1 day on IFNγ, IL-4 and IL-5 levels was more than the group treated with 1.0 mg / 1 day It was great. In contrast, IFNγ, IL-4 and IL-5 levels were decreased in the dexamethasone treated group compared to the control or drug treated group.

Example 14

Analysis of Immune Suppression. Several compounds were characterized for their ability to respond to immune responses. This protocol examined the immune effects of compounds with standard immunoassays. Ovalbumin (OVA) specific immune response assays are well-established systems for measuring analog (tic and antibody mediated) immune responses. BALB / c mice were immunized with 100 μg OVA precipitated with alum (25 mg / ml) in saline, by intraperitoneal injection (200 μl total volume) on days 0 and 7. Mice (n = 5 per group) were treated with compounds daily for 20 days (40 mg / kg via oral feeding, about 1 mg / animal) for 20 days. On day 20, blood was collected and tested for antibody titers against OVA by ELISA. Compounds tested were 3β, 16α-dihydroxy-17-oxoandrostane, 16α-bromoepiandrosterone, 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, 3β, 16α- Dihydroxyandrostan-17-oxime, 17β-aminoandrost-5-ene-3β-ol and 3α, 16α, 17β-trihydroxyandrostan. None of the compounds significantly inhibited immunity and the OVA antibody titers were similar to those of the vehicle control.

Example 15

Lowering blood sugar and reducing insulin resistance. The hypoglycemic effect and the alleviating effect of insulin resistance were measured in a diabetic db / db mouse model, a model of human diabetes and insulin resistance. In this study, approximately 8 to 10 week old db / db C57BL / Ks mice were divided into 10 groups and then 17α-ethynylandrost-5-ene via vehicle control (drug free) or oral feeding. -3β, 7β, 17β-triol were treated. Compound is 20 mg / kg / day twice daily (twice at a dose of 10 mg / kg administered once a day), 40 mg / kg / day (20 mg administered once a day) up to 28 days at 80 mg / kg / day (2 doses at 40 mg / kg once daily). During the dosing period, blood glucose levels were measured twice a week during the dosing period using a small amount of blood (slightly cut in the tail) to measure blood glucose levels via a glucometer. At certain times during the dosing period (day 28, day 14), an oral glucose tolerance test (OGTT) was performed using a standard oral dose of 1 g / kg glucose (approximately 40 mg in 40 mg mice), followed by blood glucose Changes in values were quickly observed 15, 30, 60 and 120 minutes after glucose administration. In the drug treated group, blood glucose levels were found to be reduced approximately 40% in db / db mice. Blood glucose in the vehicle control group was approximately 380 mg / dL and <230 mg / dL at least 10 days after administration in the drug-treated group. Treatment with drug at 80 mg / kg bid for 28 days resulted in blood glucose at <400 mg / dL in the drug-treated group at approximately 400 mg / dL 30 minutes after oral glucose administration observed in vehicle-treated animals. Significantly reduced.

Example 16

Treatment of hyperglycemia in feeding induced obesity (DIO) mice. The effects of drugs that enhance peripheral sensitivity to insulin can be studied in mouse models in which the state of insulin resistance has been obtained by feeding animals at least 6 weeks fat-rich foods (60% of total calorie intake). This model is described, for example, in JN Thupari et al., Proc. Natl. Acad. Sci . USA, 99 (14): 9498-9502, 2002, H. Xu et al., J. Clin.Invest., 112: 1821-. 1830, 2003, H. Takahashi et al., J. Biol. Chem ., 278 (47): 46654-46660, 2003). Under these feeding conditions, the mice gained weight (+35 g) and were intolerant of glucose, indicating a significant delay in the clearance time of orally administered glucose during the standard OGTT (oral glucose tolerance test).

For the study, approximately 4 weeks old animals were divided into groups of 10 animals each and then treated with vehicle control (no drug treatment), or via oral feeding 17α-ethynylandrost-5-ene- Treatment with 3β, 7β, 17β-triol. 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol was administered at 20 mg / kg, 40 mg / kg or 80 mg / kg twice a day for up to 28 days. OGTT was performed on days 14 and 28 of the dosing period. In the DIO-model of insulin resistance, 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol markedly reduced glucose intolerance compared to vehicle control mice, which was a significant improvement in OGTT blood glucose. Appears as This finding indicates that treatment with 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol increased peripheral insulin resistance or absorption, which improved glucose intolerance in these animals.

Example 17

A treatment protocol similar to that described in Example 15 was performed using db / db mice younger than the animals described in Example 15. The animals (n = 8 to 10 per group) were treated with 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol or vehicle, 40 mg / kg / day (20 mg / day once per day). Two doses per day were administered via oral feeding twice a day) and 80 mg / kg / day (two 40 mg / kg doses per day). At the start of dosing, the animals were 6 weeks old and had no elevated blood glucose levels or hyperglycemia. For the onset and rate of progression of hyperglycemia in the animals, administration was continued with vehicle or drug for 32 days to determine the effect of treatment. In the control group, the onset of hyperglycemia was observed 25 days after administration, and then steadily worsened, ie, blood glucose levels continued to increase until the end of the 32 days of administration from normal to hyperglycemic levels. In contrast, in the drug treatment group, blood glucose levels did not increase above the normal level until the end of the 32-day administration period, indicating that drug treatment delayed the onset of hyperglycemia during the protocol.

When 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol was administered to 8-week-old male diabetic db / db mice, high blood glucose levels were significantly lowered to baseline blood glucose, 40 mg / kg. In treatment twice a day in the dosing group, this effect became more pronounced after 10 days of administration and lasted 18 more days. In younger 6-week-old male db / db mice, treatment with 40 mg / kg of 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol was vehicle treated 25 days after the animals were administered. It completely blocked entry into the hyperglycemic state observed in the group. Treated animals maintained blood glucose levels comparable to lean db / + littermates. In addition, the results of OGTT performed in treated animal models showed significant alleviation of glucose intolerance compared to vehicle control animals.

Example 18

Blood glucose drop in 8-week-old db / db diabetic mice. Hyperinsulinemia-normal blood glucose clamp protocol was performed to measure insulin sensitivity in vivo. In this procedure, glucose was injected to maintain normal blood glucose or fixed normal blood glucose levels (about 180 mg / dL) while administering insulin to increase insulin concentration. The glucose infusion rate (GIR) required to maintain normal blood glucose showed insulin action in these animals. The purpose of this protocol was to reduce 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol and androst- to reduce systemic insulin resistance and improve arrest glucose in the hyperinsulinemia-normal blood glucose clamp model. To characterize the ability of 5-ene-3β, 7β, 16α, 17β-tetrol. Skeletal muscle and liver insulin sensitivity and tissue specific glucose uptake were also evaluated. Animals were administered daily via oral feeding for 14 days. On day 10 of administration, the catheter was inserted into the carotid and jugular veins. On the day of clamp (day 14), the compound was administered at 7:30 am.

Body weight and glucose concentrations were measured on days 0, 7 and 14 of treatment. On day 14, the hyperglycemic hyperinsulinemia clamp was performed. The food was removed at 7:30 min and injected continuously at 10:30 [3- ³ H] -glucose (0.05 mCi / min). Baseline blood samples were taken at 12:50 (10 minutes before) and the normal blood glucose hyperinsulinemia clamp was initiated by administering 10 mU / kg / min of insulin at 1 (0 minutes). At varying rates glucose was injected into the clamp, resulting in a glucose concentration of about 180 mg / dl. A bolus of [ ¹⁴ C] -2 deoxyglucose was injected at the end of the study to measure tissue specific glucose uptake. Plasma ¹⁴ C 2-deoxyglucose was measured at 122, 125, 130, 135, 145 minutes. Sodium pentobarbital was then anesthetized by intravenous injection, then the selected tissue was removed, immediately frozen in liquid nitrogen and stored at −70 ° C. until analysis.

The analysis was performed as follows. Plasma samples were removed with Ba (OH) ₂ (0.3 N) and ZnSO ₄ (0.3 N), dried and the radiation measured by a scintillation counter (Packard TRICARB 2900 TR, Meriden, CT). It was. Frozen tissue samples were homogenized in 0.5% perchloric acid, centrifuged and then neutralized. A portion of the supernatant was directly counted and the radiation measured for [ ₁₄ C] DG and [ ¹⁴ C] DGP. The second aliquot was treated with Ba (OH) ₂ and ZnSO ₄ to remove ¹⁴ C DGP, allowing any residue to enter the glycogen, and then measuring radiation from free [ ¹⁴ C] DG (2). It was. [ ¹⁴ C] DGP was calculated as the difference between the two aliquots. Accumulation of [ ¹⁴ C] DGP was normalized to tissue weight and tracer bolus. Rg, the tissue specific glucose uptake coefficient, was calculated as previously described in EW Kraegen et al., Am. J. Physiol ., 248: E353-E362, 1985. Total body glucose turnover was calculated as the ratio of ³ H glucose infusion rate (dpm / kg / min) and arterial plasma glucose specific activity (dpm / mg). Endogenous glucose production rate was calculated as the difference between body glucose turnover and external glucose infusion rate (RN Bergman et al., Endocr. Rev. , 6: 45-86, 1985). Treatment groups are summarized in the table below.

group process Dosage and Dose Concentration N A-vehicle control * Vehicle 8 ml / kg, po, bid qd at 13 days, 14 days 8 ml / kg 10 B- Compound 1 ** 40 mg / kg, po, bid qd at 13 days, 14 days 4 ml / kg of 10 mg / ml stock in vehicle 10 C- Compound 1 ** 80 mg / kg, po, bid qd at 13 days, 14 days 8 ml / kg of 10 mg / ml stock in vehicle 10 D- Compound 2 ** 40 mg / kg, po, bid qd at 13 days, 14 days 4 ml / kg of 10 mg / ml stock in vehicle 10 E-positive *** control 25 mg / kg, po, bid qd at 13 days, 14 days 5 mg / ml in water to 5 ml / kg + 1% CMC 10  Vehicle: 30% sulfobutyl ether in water (20 mg / ml of drug in solution for group BD) Compound 1: 17α-ethynylandro-5-ene-3β, 7β, 17β-triol Compound 2: Androst-5-ene-3β, 7β, 16α, 17β-tetrol *** rosiglitazone maleate (31493r, AApin Chemical Limited (UK) CMC-carboxymethyl cellulose (medium grade, C4888, Sigma)

Insulin dose was 10 mU / kg / min. In normal animals, this insulin dose was required to inject about 90 mg / kg of glucose to maintain the clamped glucose level at about 150 mg / dl. Mean glucose requirement in all treatment groups was about 50% of normal value. The results indicate that both 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol and androast-5-ene-3β, 7β, 16α, 17β-tetrol reduce the glucose infusion rate compared to the vehicle control. Increased, indicating an improvement in insulin activity in groups B, C, D and E.

Using a 3 ³ H glucose tracer, hepatic glucose production rate was calculated during the baseline time and the ability of insulin to inhibit glucose production during the clamp period. In severe insulin resistant animals, endogenous glucose production rate was reduced by about 50% with the insulin dose used. Insulin in groups C, D and E completely inhibited endogenous glucose production (p <0.05), indicating an improvement in hepatic insulin action.

To measure peripheral insulin action, tissue specific glucose uptake in the hyperglycemic hyperinsulinemia clamp was measured using ¹⁴ C-2-deoxyglucose. ^A bolus of ¹⁴ C-2-deoxyglucose was applied for 120 minutes. Tissue was collected after 25 minutes. Tissues were analyzed for total accumulation of ¹⁴ C-2-deoxyglucose phosphate. In this protocol, brain glucose uptake was not affected by the best treatment regimen and served as an internal control. This result shows that brain glucose uptake is similar between all groups. In the heart and diaphragm, glucose uptake was higher in the drug treated group compared to the vehicle control. Androst-5-ene-3β, 7β, 16α, 17β-tetrol and rosiglitazone were more effective for augmenting muscle glucose uptake in gastrocnemius muscle (p <0.05). In the white vastus muscle (which is a non-oxidative muscle group), no difference was detected except between androst-5-ene-3β, 7β, 16α, 17β-tetrol and rosiglitazone.

Example 19

Rats were fed free for 6 days with standard laboratory food, i.e., containing 0.45% w / w of androst-5-ene-3β, 7β, 17β-triol, followed by phosphoenolpyruvate carboxykinase ( Hepatic tissue was analyzed for 6 days for liver levels of "PEPCK") and 11β-hydroxysteroid dehydrogenase ("11β-HSD"). Control animals were fed normal and tested for 6 days for PEPCK and 11β-HSD levels for 6 days by measuring messenger RNA (mRNA) by RT-PCR. Control and treatment animals were fed with water freely. Compound administration during 6-day feeding was found to reduce the levels of 11β-HSD type 1 (“11β-HSD1”) and PEPCK in liver tissue as shown below. PPARα mRNA levels in these animals were not affected by feeding androst-5-ene-3β, 7β, 17β-triol.

11β-HSD1 mRNA PEPCK mRNA PPARα mRNA Control (no compound) 100% 100% 100% Androst-5-ene-3β, 7β, 17β-triol 45% 30% 105%

In another study, administration of compound 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol to mice was found to reduce the expression of 11β-HSD1 in osteoblasts by 50%. This is consistent with the finding that the compound has a bone-sparing effect in mice treated with dexamethasone, glucocorticoids that induce bone loss in vivo.

In another study, from adipose tissue around the gonads from db / + or diabetic db / db mice treated with 20 mg / kg of 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol Isolate whole RNA from quantitative RT using primers specific for monocyte chemotractant protein-1 (MCP-1) using iCycler iQ multicolor real time-detection system (Bio-Rad) / PCR was performed. RNA expression levels were normalized to vehicle control. The compound was found to reduce the level of monocyte chemoretractant protein-1 (MCP-1) by about 50%. For this study, vehicles were also administered to controls of age matched lean heterozygous db / + mice (n = 7).

17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androst-5-ene-3β, 7β, 16α, 17β-tetrol, androst-5-ene-3β, 7α, 16α , 17β-tetrol, androst-5-ene-3α, 7β, 16α, 17β-tetrol, androast-5-ene-3β, 4β, 16α, 17β-tetrol, androst-5-ene-3α Monoesters or diesters of such compounds, such as, 4β, 16α, 17β-tetrol or compounds containing one or two acetate or propionate esters in the 3- or 17-positions, are also PEPCK or 11β- In a similar manner as to HSD type 1 or 11β-HSD type 2 or the like or their ability to reduce the level or activity of 11β-HSD, such as in hepatocytes or cells derived from the liver, or other tissues or other cells In, for example, kidneys, muscles, bone tissues or cells, in adipose tissues or cells, or in CNS tissues or cells, for example neurons Articles examined in Trang.

Example 20

Inhibition of development of CD4 ⁺ CD25 ⁺ T regulatory cells or their activity in vivo. Purified CD4 ⁺ CD25 ^- T cells (5 × 10 ⁶ cells) from congenic B6.SJL mice (CD45.1) per group were adapted to each mouse of five B6 mice (CD45.2). Delivered. Purified CD4 ⁺ CD25 ⁻ T cells were obtained by fluorescence activated cell sorting (FACS) of at least two donor cells. Vehicles (0.1% carboxymethylcellulose, 0.9% saline, 2% tween 80, 0.05% phenol) or 16α-bromoepiandrosterone (in 100 ml of vehicle, 1 mg / animal / day) in vehicle were CD45.1 donors The cells were injected subcutaneously before delivery from the animals and the injection continued every 14 days. Thymus, lymph nodes and spleen were collected from animals on day 15. Thymus, lymph node and spleen samples were collected separately and cells were labeled with fluorescent antibodies that bind to CD4, CD25, CD103 or Foxp3. Cells were then analyzed by flow cytometry to determine the number of different cell types. Remaining lymph nodes and spleen cells from each treatment group were collected, pre-enriched for CD4 ⁺ CD25 ⁺ cells, followed by host (endogenous CD45.2 cells) and donor cells (in vivo for 15 days). after CD4 ⁺ CD25 ^- was analyzed with respect to the CD4 ⁺ CD25 ⁺ cells induced from CD45.1 ⁺ CD25 ⁺ phenotype in the transition to donor phenotype CD4). Cells were analyzed by cell sorter. To test regulatory functions, various numbers of purified converted CD45.1 or endogenous CD45.2 CD4 ⁺ CD25 ⁺ cells were irradiated with 2000 CD4 ⁺ CD25 ⁻ responder cells and 1 × 10 ⁵ irradiated spleen as antigen presenting cells. Cells, and incubated with 0.5 mg / ml of anti-CD3 antibody. Fresh CD4 ⁺ CD25 ⁺ cells were used as a control. The extent of proliferation was measured by measuring ³ H-thymine uptake 4 days after the start of the culture.

The test results indicated that the number of donor CD45.1 CD4 ⁺ CD25 ⁺ Tregs cells in the spleen of drug-treated animals was less than the number of CD45.1 CD4 ⁺ CD25 ⁺ Tregs cells in the spleen from vehicle control animals. . The mean vehicle control CD45.1 CD4 ⁺ CD25 ⁺ cell number was 1.97 × 10 ⁵ cells compared to 0.62 × 10 ⁵ CD45.1 CD4 ⁺ CD25 ⁺ cells, the average number from animals treated with drugs.

The number of endogenous CD45.2 CD4 ⁺ CD25 ⁺ Tregs cells in the spleen from animals treated with drugs was less than the number of CD45.1 CD4 ⁺ CD25 ⁺ Tregs cells in the spleen from vehicle control animals. The mean vehicle control CD45.2 CD4 ⁺ CD25 ⁺ cell number was 9.54 × 10 ⁶ cells compared to the mean 5.49 × 10 ⁶ CD45.2 CD4 ⁺ CD25 ⁺ cells treated with the drug.

The mean endogenous CD45.2 CD4 ⁺ CD25 ⁺ cells in the thymus of the vehicle control animals were 3.10 × 10 ⁵ compared to 1.59 × 10 ⁵ in the drug treated animals.

The ratio of donor CD45.1 CD4 ⁺ CD25 ⁺ CD103 ⁺ cells to the total donor CD4 ⁺ CD25 ⁺ cells in the spleen of the animal treated with the drug was compared to the total donor CD4 ⁺ CD25 ⁺ cells in the spleen from the vehicle control animal. CD45.1 was lower than the number of CD4 ⁺ CD25 ⁺ CD103 ⁺ Treg cells. The proportion of endogenous CD45.2 CD4 ⁺ CD25 ⁺ CD103 ⁺ Treg cells was approximately the same in the spleen from vehicle control animals (13.85%) compared to animals treated with drug (13.40%). The mean donor CD45.1 CD4 ⁺ CD25 ⁺ CD103 ⁺ cells for the vehicle control group were 29.06% compared to 9.63% on average in the animals treated with the drug. Embryonic CD103 surface antigen is expressed in activated Treg cells. This indicates that the relative proportion of activated CD45.1 CD4 ⁺ CD25 ⁺ cells is lower in drug-treated animals than in the vehicle control, which is consistent with the inhibition of Treg cell activity on donor cells in vivo.

Suitable variations of this protocol include: (1) using more donor CD4 ⁺ CD25 ^- T cells per animal, for example 1 x 10 ⁶ / animal, 1.5 x 10 ⁶ / animal or 2 x 10 ⁶ For use as an animal, (2) varying the daily dosage of the drug, (3) varying the route of administration of the drug, (4) using other compounds as the drug and (5) additional animal Including a group, for example, including a group receiving an anti-inflammatory agent or an immunosuppressant glucocorticoid such as dexamethasone or cortisol. Some of these modifications can be applied to the protocol in Example 3 or in some citations.

Example 21

Increase in CD4 ⁺ CD25 ⁺ T regulatory cells or measure their in vivo activity. Collagen-induced arthritis animal model (H. Offner et al., Clin. Immunol ., 110: 181-190, which has previously described compound 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol) 2006) was administered in substantially the same manner.

DBA / 1Lac / J mice were used for the study. Mice were purchased from Jackson Laboratories (Bar Harbor, Harbor, Mass.) And bred according to laboratory guidelines. Eight-week-old mice with 200 μg of bCII emulsified in small size II collagen (bCIII) 1: 1 with CFA containing 200 μg Mycobacterium tuberculosis (100 μL; Difco, Detroit, MI) Collagen-induced arthritis (CIA) was induced by immunization. Antigen was injected into the skin at the base of the tail. Animals were observed for disease development and progression after immunization over 4-7 weeks. Arthritis severity was assessed by scoring each foot according to the following score: 0 = redness or edema; 1 = slight swelling of the ankle or redness at the foot; 2 = edema and inflammation progress and redness from the ankle to the middle of the foot; 3 = edema and inflammation throughout the foot; 4 = edema and inflammation throughout the foot, including the toenails.

After immunization, at the onset of observable clinical disease, mice were treated via oral feeding with 40 mg / kg / day of drug in vehicle (starting about 26-27 after immunization). The vehicle used for the protocol was 30% cyclodextrin-sulfobutylether in water. Cyclodextrin-sulfobutylether was commercially available (Captisol ^™ available from cydexinc.com). The drug formulation was 20 mg drug / mL in the vehicle.

Results obtained from drug-treated animals showed that Foxp3 ⁺ and CD4 ⁺ throughout the spleen cells, as shown below, as a result of administration of 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol. It was shown that the frequency of Foxp3 ⁺ expressing cells was increased.

Vehicle (n = 3) Drug (n = 3) p value Full Foxp3 ⁺ 1.6% ± 0.16 2.39% ± 0.16 0.00001 CD4 ⁺ Foxp3 ⁺ 1.1% ± 0.09 1.34% ± 0.05 <0.00001

Cell sorter analysis showed an increase in Foxp3 + CD4 ⁺ cells in drug treated animals (1.4%) compared to control animals (1.0%). Foxp3 protein is associated with the differentiation or conversion of CD4 ⁺ CD25 ^- T cells into CD4 ⁺ CD25 ⁺ Treg cells, indicating that as the number of Foxp3 expressing cells increases, the development of their precursor cells to Treg cells has increased. . After immunization, mice were treated via oral feeding with 40 mg / kg / day of drug in vehicle at the onset of observable clinical disease (starting about 26-27 days after immunization). Results obtained from drug-treated animals showed that treatment with 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, Foxp3 ⁺ and CD4 ⁺ Foxp3 ⁺ in whole splenocytes, as shown below. It was shown to increase the frequency of expressing cells. Consistent with this was a statistically improved clinical score in animals treated with drugs compared to vehicle controls 44 to 49 days after immunization. From day 39 to day 49, vehicle control animals had an average clinical score of about 6.8 to 8, whereas drug-treated animals gradually decreased with a maximum mean clinical score of about 5 on day 34 and then on day 49 The average score was about 3 points. These results show that this compound slows arthritis progression and reduces maximal severity compared to vehicle control animals.

Example 22

Synthesis of the compound is as follows.

Androst-5-ene-3β, 7β, 16α, 17β-tetrol (7) .

5-Androsten-3β, 16α-diol-17-one diacetate (3). 16α-bromodehydroepiandrosterone 2 was prepared by refluxing DHEA (1) in methanol using copper (II) bromide. 15 g of 2 (40.8 mmol) compound in pyridine (129 ml) and water (309 ml) were added to 120 ml of aqueous 1N sodium hydroxide solution and the mixture was stirred in air for 15 minutes. The reaction mixture was poured into ice / water containing excess hydrochloric acid as saturated with sodium chloride. The crude product was filtered off, washed with water until neutralized and dried in vacuo on anhydrous calcium chloride at 55-60 ° C. Recrystallization of methanol gave 8.21 g of 16α-hydroxy-DHEA (Mp 194.4-195.1 ° C.). This product was then converted to diacetate (3) by treatment with excess acetic acid in pyridine and purified by flash chromatography.

5-androsten-3β, 16α-diol-7,17-dione (5). To a solution of 3 (20.1 g, 51.7 mmol) in benzene containing celite (60 g), pyridinium dichromate (75 g) was added to 22 ml of 70% tert-butyl hydrogen peroxide. After 2 days of stirring at room temperature, diethyl ether (600 mL) was added and the precipitate was filtered off and washed with ether (2 × 100 ml). The residue was purified by flash chromatography (60% ethyl acetate in hexanes) and recrystallized to give 16.0 g (39.8 mmol, 77%) of 5 in the form of a prism. Mp 205.6-206.2 ° C.

5-androsten-3β, 7β, 16α, 17β-tetrol (7). To a solution of 5 (10.0 g, 24.8 mmol) in dichloromethane (75 ml) and methanol (255 ml) at 0 ° C., 1.5 g of sodium borohydride were added and the mixture was stirred at 0 ° C. for 1 hour. After quenching with acetic acid (3.5 ml), the reaction mixture was partitioned between dichloromethane and water. The organic layer was concentrated to a mixture of 7α and 7β diacetate tetrols. The mixture was purified by flash chromatography and HPLC to give 2.90 g of 7β-epimer (9.5 mmol, 38%). Mp 216.8-220.8 ° C. Saponification with 1 N sodium hydroxide (60 ml) in methanol for 2 days at room temperature and purification by HPLC gave 7 (1.41 g, 4.4 mmol, 46%) in the form of a fine needle from an aqueous acetonitrile solution. . Mp 202.1-206.4 ° C; [a] D +1.35 (methanol, c = 1). Selected ¹ H NMR peak (CD ₃ OD): d 0.77 (s, 3H), 1.01 (s, 3H), 3.39 (d, 1H), 3.46 (m, 1H), 3.74 (t, 1H), 4.04 (m , 1H), 5.55 (dd, 1H).

3α, 7α, 17β-triacetoxyandrost-5-ene-16α-ol (8), androast-5-ene-3α, 7α, 16α, 17β-tetrol (9) .

16α-bromo-5-androsten-3α-ol-17-one (2) . A solution of 5-dehydroandrosterone ( 1 ) (17.8 g, 61.7 mmol) in methanol (1.35 L) was refluxed with copper (II) bromide (36.4 g, 163 mmol) with stirring for 19 h. To the quenched reaction mixture was added water (1.35 L) and dichloromethane (1.5 L). The organic layer was filtered through anhydrous sodium sulfate, and the product crystallized from methanol in the form of a fine needle (16.7 g, 45.5 mmol, 74%). Mp 195-207 ° C.

3α, 16α-diacetoxy-5-androsten-17-one (4). To a 2 (12.0 g, 32.7 mmol) solution in pyridine (1.032 L) and water (0.247 L) was added an aqueous solution of 1N sodium hydroxide (90 ml) in air, then the mixture was stirred at room temperature for 15 minutes. The reaction mixture was added to an ice / water mixture containing 1.2 L 1N hydrochloric acid. The mixture was saturated with sodium chloride and then extracted with ethyl acetate (2 × 1 L). The combined organic layers were washed with brine (250 ml), filtered over anhydrous sodium sulfate and concentrated. The crude 5-androsten-3α, 16α-diol-17-one ( 3 ) was treated with excess acetic anhydride in pyridine overnight at room temperature and then purified by column to give 4 (7.46 g, 19.2 mmol, 59%) was produced in prismatic form from methanol. Mp 172.7-173.7 ° C.

5-androsten-3α, 16α, 17β-triol 3,16-diacetate (5). To a solution of endiolone diacetate 4 (7.46 g, 19.2 mmol) in dichloromethane (45 ml) and methanol (120 ml) was added sodium brohydride (950 mg) at 0 ° C. This solution was stirred for 1 h at 0 ° C. Excess acetic acid was added to the reaction mixture and then partitioned between dichloromethane and water. The organic layer was filtered over anhydrous sodium sulfate and then concentrated to give a mixture of 17α (small amount) and 17β (large) epimer. This mixture was purified by flash chromatography (25% ethyl acetate in hexanes), resulting in 6.1 g (15.6 mmol, 81%) of 17β Epimer 5 . Mp 126.9-128.6 ° C. Triacetate 6 prepared from 5 was treated with excess acetic anhydride in pyridine overnight at room temperature and then purified by column to give 6.0 g (13.9 mmol, 89%).

5-androsten-3α, 16α, 17β-triol-7-one triacetate (7). Benzene (255 Triacetate 6 (6.0 g, 13.9 mmol) solution in ml) was treated with celite (25.5 g), pyridinium dichromate (31.5 g) and 70% tert -butyl hydrogen peroxide (9.0 mL) and room temperature Stirred for 19 hours. Anhydrous diethyl ether (255 ml) was added and the reaction mixture was cooled in a cold water bath for 1 hour. The resulting solid was filtered off and washed with ether (2 × 50 mL). The combined organic portions were concentrated and then purified by flash chromatography (29% ethyl acetate in hexanes), resulting in 3.45 g of 7 (7.7 mmol, 55%).

5-androsten-3α, 7α, 16α, 17β-tetrol (9). To a solution of 7 (3.45 g, 7.7 mmol) in dichloromethane (15 ml) and methanol (30 ml) was added sodium borohydroxide (1.0 g) at 0 ° C. and the solution was stirred at 0 ° C. for 2 hours. Excess acetic acid (1.5 ml) was added and then the reaction mixture was partitioned between dichloromethane and water. The organic layer was filtered over anhydrous sodium sulfate and then concentrated to yield a mixture of 7α (small amount) and 7β (large) epimer. This mixture was saponified in methanol (100 ml) using 1 N sodium hydroxide (60 ml) overnight at room temperature. The crude tetrol was recovered by partitioning the saponification mixture between ethyl acetate and brine. Epimers were separated by HPLC to yield 220 mg of 9 (0.68 mmol, 9%). Mp 243-248.3 ° C. ). Selected ¹ H NMR peak (CD ₃ OD): δ 0.77 (s, 3H), 1.02 (s, 3H), 2.11 (m, 1H), 2.57 (m, 1H), 3.34 (s, 1H), 3.44 (d , 1H), 3.70 (br t, 1H), 4.04 (m, 2H), 5.55 (dd, 1H). The epimer of 8 was separated by HPLC to obtain purified 8 and its 7β-acetate epimer.

Androst-5-ene-3β, 7β, 11β, 17β-tetrol-3β-acetate (8), androast-5-ene-3β, 7β, 11β, 17β-tetrol (9), androast-5 -N-3β, 7β, 17β-tetrol-3β-acetate-11-oxime (10).

I : 2.8 g of p-TsOH was added to a 1 (4 g) solution in 150 ml Ac ₂ O, and 700 ml cold water was added overnight at room temperature, while stirring for 1 hour until becoming a solid form, and filtered. , White solid product 2 (4.55 g) was produced.

II : To a 1.5 g NaBH ₄ solution in 35 ml EtOH and 5 ml MeOH was added 2 (1.2 g) solution in 30 ml EtOH and 10 ml chloroform at 0 ° C. The solution was continued stirring at 0 ° C. for 2 hours and then stirred at room temperature for 2 hours. At this time 4 ml acetic acid was added to quench NaBH ₄ and then 50 ml water was added. The product was separated by extraction three times with 50 ml of EtoAc and the solvent was removed in vacuo to afford the crude product. Purification via column chromatography yielded 3 product (250 mg).

III: To a 3 (200 mg) solution in 8 ml MeOH, add 0.36 g H ₅ IO ₆ solution in 2 ml water, stir at room temperature for 1 hour, remove the solvent in vacuo, add water and extract dichloromethane Was performed. Purification using column chromatography yielded product 4 (60 mg).

IV : To a solution of 4 (0.4 g) in 5 ml pyridine, 0.5 ml acetetyl chloride was slowly added at 0 ° C., continued stirring at 0 ° C. for 15 minutes and then at room temperature for 30 minutes. The reaction was quenched by addition of 20 ml water, extracted three times with 15 ml of EtoAc and then washed with 1N HCl, NaHCO ₃ , brine and then dried over Na ₂ SO ₄ . Concentration in vacuo gave 5 (520 mg).

V : 3 ml 70% t-BuOOH was slowly added to 5 (0.5 g) and 0.13 g CuI solution in 15 ml acetonitrile and stirred for 1 hour, followed by stirring at 50 ° C for 2 hours. 12 ml 10% Na ₂ S ₂ O ₅ solution was added followed by extraction with EtOAc, dried over Na ₂ SO ₄ , then the solvent was removed and run on a column to give 6 (80 mg).

VI: To a 6 (50 mg) solution in 1.5 ml THF and 3 ml MeOH, add 260 mg CeCl ₃ .7H ₂ 0, then slowly add 75 mg NaBH ₄ at 0 ° C., stir for 30 minutes, and then 0.5 mL 1N HCl And 5 ml water were added, extracted three times with 5 ml of EtoAc, dried over Na ₂ SO ₄ , and the solvent was removed to give 7 (49 mg).

VII: To a 300 mg NaBH ₄ solution in 4 ml EtOH and 1 ml MeOH was added 7 (40 mg) solution in 0.5 ml EtOH and 0.5 ml chloroform, stirred at 0 ° C. for 8 hours and then frozen overnight. Acetic acid was added to quench the reaction, water was added, and then EtoAc extracted to give 8 (30 mg). mp> 250 ° C .; ¹ H NMR (CD ₃ OD) δ 0.86 (s, 3H), 1.35 (s, 3H), 1.95 (s, 3H), 3.55 (t, 1H, J = 7.5 Hz), 3.71 (dd, 1H, J = 7 Hz, J = 2.5 Hz), 4.32 (d, 1H, J = 2.7 Hz), 4.55 (m, 1H), 5.21 (s, 1H)

VIII : To 8 (30 mg) solution in 1 mL MeOH, 50 mg NaOH solution in 0.25 ml water was added. After stirring at 50 ° C. for 15 minutes, 1 mL of 1N HCl and 5 ml of water were added, followed by extraction three times with 5 mL of EtoAc, and removal of the solvent gave 9 (20 mg). mp 170 to 172 ° C .; ¹ H NMR (CD ₃ OD) δ 0.95 (s, 3H), 1.32 (s, 3H), 3.41 (m, 1H), 3.51 (t, 1H, J = 8.0 Hz), 3.78 (dd, 1H, J = 7.1 Hz, J = 2.5 Hz), 4.31 (d, 1H, J = 2.5 Hz), 5.15 (s, 1H)

IX: To a 29 mg NH ₂ OH.HCl and 17 mg NaOH solution in hot 1 ml EtOH, a 9 (50 mg) solution in hot 1 ml EtOH was added and refluxed at 100 ° C. for 2 hours, after which the salt was filtered After recrystallization in EtOH / H ₂ 0, 10 (40 mg) was produced. mp> 250 ° C .; ¹ H NMR (CD ₃ OD) δ 0.72 (s, 3H), 1.02 (s, 3H), 2.03 (s, 3H), 3.86 (t, 1H, J = 8.5 Hz), 4.08 (dd, 1H, J = 8.0 Hz, J = 2.6 Hz), 4.60 (m, 1H,), 5.19 (s, 1H)

17α-methylandrost-5-ene-3β, 17β-diol-3β-acetate-7,11-dione (7), 17α-methylandrost-5-ene-3β, 7β, 17β-triol-3β- Acetate-11-one (8), methylandrost-5-en-3β, 7β, 17β-triol-11-one (9) .

I : To 1 (4 g) solution in 150 ml Ac ₂ O, 2.8 g of p-TsOH was added overnight at room temperature, stirred for 1 hour while adding 700 ml cold water, and the solid was filtered to give white product 2 ( 4. 55 g) was produced.

II : To a 1.5 g NaBH ₄ solution in 35 mL EtOH and 5 mL MeOH, 2 (1.2 g) solution in 30 mL EtOH and 10 mL chloroform were added slowly at 0 ° C., 2 h at 0 ° C., 2 h at room temperature. After continued stirring, 4 ml of acetic acid was added to quench NaBH ₄ , 50 ml of water was added, followed by extraction three times with 50 mL of EtoAc, and the solvent was removed to give a crude product. 3 (250 mg) was produced in the column.

III: To 3 (200 mg) solution in 8 ml MeOH, add 0.36 g H ₅ IO ₆ solution in 2 ml water, stir for 1 hour at room temperature, remove solvent, add water, extract with DCM The column was turned over to give product 4 (60 mg).

IV : To 4 (250 mg) solution in 1.5 mL THF and 3.5 mL ether at -78 ° C. under N ₂ , slowly add 1 mL 22% MeMgCl in THF, stir at −78 ° C. for 1.5 hours and then at room temperature for 1 hour. After stirring, the mixture was refluxed at 75 ° C. for 1 hour. 4 mL 1N HCl and 10 mL water were added at 0 ° C. Extraction with EtoAc and removal of solvent gave 249 mg of crude. The column was turned to give 5 (46 mg).

V : To a 5 (1.0 g) solution in 15 ml pyridine, 1.1 ml acetic acid was slowly added at 0 ° C., stirred at 0 ° C. for 15 minutes, then stirred at room temperature for 30 minutes, 50 ml water was added and 50 mL of EtoAc Extracted three times, washed with 1N HCl, saturated NaHCO ₃ , brine and dried over Na ₂ SO ₄ . Removal of the solvent gave 6 (1.02 g).

VI : To 6 (1.0 g) and 0.3 g CuI solution in 40 ml acetonitrile, 6 mL 70% t-BuOOH was slowly added, stirred at room temperature for 1 hour and then at 50 ° C. for 2 hours. 24 mL 10% Na ₂ S ₂ O ₅ solution was added, extracted with EtoAc, dried over Na ₂ SO ₄ , solvent removed and run on column, to give 7 ( 285 mg). mp> 250 ° C .; ¹ H NMR (CD ₃ Cl) δ 0.82 (s, 3H), 1.29 (s, 3H), 2.05 (s, 3H), 4.70 (m, 1H,), 5.75 (s, 1H)

VII : To a 7 (45 mg) solution in 1.5 mL THF and 3 mL MeOH, 150 mg CeCl ₃ .7H ₂ 0 was added, 30 mg NaBH ₄ was slowly added at 0 ° C., then stirred for 10 minutes, 0.5 ml 1N HCl and 5 ml water were added, extracted three times with 5 mL of EtoAc, dried over Na ₂ SO ₄ , and the solvent was removed, resulting in 8 (41 mg). mp 108-110 ° C; ¹ H NMR (CD3OD) δ 0.725 (s, 3H), 1.25 (s, 3H), 2.01 (s, 3H), 4.02 (dd, 1H, J = 8.2 Hz, J = 2.4 Hz), 4.53 (m, 1H ,), 5.29 (s, 1H)

VIII : To 8 (22 mg) solution in 1 mL MeOH, 23 mg NaOH solution in 0.1 mL water was added. Stir for 10 min at 50 ° C., add 1 mL of 1N HCl, add 5 mL of water, and extract three times with 5 mL of EtoAc. Removal of the solvent gave 9 (10 mg). ¹ H NMR (CD3OD) δ 0.75 (s, 3H), 1.24 (s, 3H), 3.41 (m, 1H), 3.99 (dd, 1H, J = 8.2 Hz, J = 2.5 Hz), 5.23 (s, 1H )

17α-ethynylandrost-5-ene-3β, 7β, 16α, 17β-tetrol (8), 17β-ethynylandrost-5-ene-3β, 7β, 16α, 17α-tetrol (9),

I : To 1 (1.0 g) and 0.56 g imidazole solution in 15 ml DMF, 1.24 g of TBDMS-Cl was added overnight at room temperature, 50 ml of water was added, which gave a solid which was filtered to give a white solid product 2 (1.75 g) was produced.

II : To 2 (1.64 g) solution in 50 ml THF cooled to -78 ° C, 2.3 ml LDA was added, after 30 minutes 0.62 ml TMSCl was added slowly, stirred at -78 ° C for 30 minutes, then at room temperature Allow to warm then stir for 1 hour. TLC was seen, RXN was complete, extracted twice with 150 ml of ether, then washed with water and brine, dried over Na ₂ SO ₄ , resulting in yellow product 3 (1.87 g).

III & IV : To 3 (10 g) solution in 250 ml THF cooled to -20 ° C., 4.2 g of m-CPBA was added and stirred for 3 hours, forming 4, which was slowly added 250 ml MeOH. After stirring at -20 ° C for 30 minutes, 200 ml Na ₂ SO ₃ solution was slowly added at -20 ° C and stirred for 1 hour. After warming to room temperature, extraction with 150 ml of ether three times, washing with saturated NaHCO ₃ , brine and drying over Na ₂ SO ₄ gave 11 g of unpurified product which was spared in the product. To remove CPBA, a short column, ie, five times with 50 ml of 100% Hex, then five times with 100 ml of 50% Hex / EtoAc, collected crude product 5 (8.5 g).

V: To a 10 g 90% lithium acetylide ethylene diamine complex solution in 250 ml dry THF was added a 5 (5 g) solution in 50 ml dry THF over 8 hours using a syringe pump. It was allowed to stir overnight at room temperature. 500 ml water at 0 ° C. was added, extracted three times with 150 mL of EtoAc, washed with 200 mL 0.1 N HCl, 150 mL saturated NaHCO ₃ , 100 mL brine, dried over Na ₂ SO ₄ , and the solvent was removed. 5.5 g of crude product was produced. Two isomers, 17-β-OH, 6 (1.5 g) and 17-α-OH, 7 (1.2 g), were collected and run on the column.

VI: To 6 (265 mg) solution in 4 mL MeOH and 3 mL THF, 4 mL 1N HCl was added for 1.5 h at room temperature. 10 ml saturated NaHCO ₃ was added, the organic solvent was removed at room temperature, 10 ml water was added and placed in the freezer overnight to remove water, THF was added to the solid, filtered and THF was removed. Solid product 8 (130 mg) was produced. mp 214-216 ° C; ¹ H NMR (CD ₃ OD) δ 0.92 (s, 3H), 1.06 (s, 3H), 2.99 (s, 1H), 3.42 (m, 1H), 3.72 (dt, 1H, J = 7.2 Hz, J = 2.5 Hz), .4.17 (dd, 1H, J = 8.2 Hz, J = 2.7 Hz), 5.24 (d, 1H, J = 1.0 Hz).

VII: To a 7 (500 mg) solution in 8 mL MeOH and 6 mL THF, 8 mL 1N HCl was added at room temperature for 1.5 h. Saturated NaHCO ₃ was added to neutralize the solution to pH = 8. 50 ml water was added to give a white solid, filtered, washed with water and dried in vacuo, resulting in white solid 9 (225 mg). mp> 250 ° C .; ¹ H NMR (CD ₃ OD) δ 0.90 (s, 3H), 1.06 (s, 3H), 2.75 (s, 1H), 3.42 (m, 1H), 3.72 (dt, 1H, J = 7.0 Hz, J = 2.0 Hz), .4.37 (dd, 1H, J = 8.1 Hz, J = 2.6 Hz), 5.24 (t, 1H, J = 2.0, J = 1.0 Hz).

4β-acetylandrost-5-ene- 3β , 16α, 17β-triol (7) , androast -5-ene- 3β , 4β, 16α, 17β-tetrol (Compound 8) .

Step 1: Compound 1 (24.0 g, 0.0832 mol) and copper bromide (56.0 g, 0.20 mol) in anhydrous methanol (800 ml) were refluxed for 16 hours. Most of the solvent was removed under vacuum and water (500 ml) was added. The resulting precipitate was collected by filtration and washed with water. The solid was recrystallized twice in methanol, resulting in compound 2 as a pale yellow solid (19.7 g).

Step 2: To a stirred solution of a solution of compound 2 (22.0 g, 0.060 mol) in 200 mL N, N-dimethylformamide, an aqueous 1 N sodium hydroxide solution (66 mL, 0,066 mol) was added. The reaction mixture was stirred at rt for 1 h. 1N aqueous hydrochloric acid solution (8 ml) and 400 ml water were added. The resulting precipitate was collected by filtration and washed with water. This crude product was purified by recrystallization in methanol to give compound 3 as a white solid (11.8 g).

Step 3: To a solution of compound 3 (11.8 g, 0.0387 mol) in pyridine (50 ml), acetyl chloride (11.8 g, 0.128 mol) was added at 0 ° C. The reaction mixture was stirred at 0 ° C. for 1 hour. The resulting mixture was warmed up to room temperature and stirred for 1 h more. Water was added. The precipitate was collected by filtration and washed with water. The solid was dried in vacuo, resulting in compound 4 (12.6 g), which was used without further purification.

Step 4: Lithium aluminum hydride (1.13 g, 0.030 mol) was added to a cold (0 ° C.) solution of compound 2 (3.10 g, 0.00916 mol) in 80 ml of anhydrous ether under nitrogen. The cold water bath was removed and the resulting mixture was stirred at room temperature for 0.5 hour and then refluxed for another hour. The reaction was quenched by adding 6N aqueous hydrochloric acid solution. The ether was removed under reduced pressure. The resulting solid was filtered off and washed with water. The crude product was recrystallized in methanol to give compound 5 (1.1 g) as a white solid.

Step 5: Bromine (303 mg, 3.16 mmol) was added to a solution of 5 (914 mg, 2.98 mmol) in 20 ml chloroform. The reaction mixture was stirred at rt for 20 min. The solvent was removed under reduced pressure to afford compound 6 which was used without further purification.

Step 6: Purification of 4β-acetylandrost-5-ene-3β, 16α, 17β-triol ( 7 ). Compound 6 was dissolved in 30 ml of anhydrous ether and 10 ml of anhydrous pyridine. A solution of silver acetate (1.03 g, 1 (914 mg, 2.98 mmol) in 5 ml of anhydrous pyridine was added. The reaction mixture was stirred for 0.5 h in the dark. A heavy greenish precipitate was deposited. Ether (50 ml) The precipitate was filtered off The precipitate was dried under vacuum The residue was purified by flash chromatography on silica gel eluting with 50:50 ethyl acetate: hexanes to give compound 7 (124 mg) as a white solid. ¹ H NMR data selected: (CD ₃ OD, 300 MHz): δ 5.78 (d, 1 H, J = 2.2 Hz), 5.34 (br, 1 H), 4.02 (t, 1 H, J = 4.5 Hz), 3.52 (dt, 1 H, J = 7.7 Hz, 3.0 Hz), 3.37 (d, 1H, J = 4.9 Hz), 2.03 (s, 3 H), (1.12 (s, 3H), 0.75 (s, 3H) Melting Point: 152-153 ° C.

Step 7: Preparation of androst-5-ene-3β, 4β, 16α, 17β-tetrol ( 8 ). Compound 7 (50 mg, 0.137 mmol) was dissolved in 1N aqueous sodium hydroxide solution (1 ml) and methanol (5 ml) and the resulting solution was refluxed for 1 hour. Methanol was removed in vacuo and the residue was extracted with ethyl acetate (3 × 30 ml). The combined extracts were dried over magnesium sulphate, filtered and concentrated in vacuo resulting in a solid. The crude product was purified by recrystallization in methanol to give compound 8 (23 mg) as a white solid. Selected ¹ H NMR data: (CD ₃ OD, 300 MHz): δ 5.62 (d, 1 H, J = 3.2 Hz), 4.05 (d, 1 H, J = 2.4 Hz), 4.02 (m, 1 H), 3.43 (dt, br, 1 H, J = 11.7 Hz, 3.6 Hz), 3.36 (d, 1H, J = 4.2 Hz), 1.197 (s, 3H), 0.76 (s, 3H). Melting Point: 238-241 ° C.

Androst-5-ene-3β, 4β, 7β, 17β-tetrol 12 (method 2), androast-5-ene-3β, 7β, 17β-triacetoxy-4β-ol (11) .

Step 1: To a solution of compound 9 (5.0 g, 0.0138 mol) in pyridine (20 ml) was added acetyl chloride (11.8 g, 0.128 mol) at 0 ° C. The reaction mixture was stirred at 0 ° C. for 5 hours, then most of the solvent was removed in vacuo. The remaining sludge was partitioned between ethyl acetate (80 ml) and water (20 ml). The organic layer was washed with 1N aqueous hydrochloric acid solution, saturated with aqueous sodium bicarbonate solution, dried over magnesium sulfate, filtered and evaporated to give a solid. The crude product was recrystallized in ethyl acetate and hexanes to give compound 10 (4.8 g) as a white solid.

Step 2: A solution of compound 10 (720 mg, 1.66 mmol) in dioxane (15 ml) and acetic acid (10 ml) was added selenium dioxide (185 mg, 1.66 mmol) in water (1.5 ml) and dioxane (5 ml). Added. The reaction mixture was heated at 95 ° C. for 36 hours. The mixture was cooled to room temperature, diluted with ethyl acetate and washed sequentially with water, saturated sodium bicarbonate and brine, then dried over magnesium sulfate, filtered and concentrated in vacuo to dry. The crude product was purified by flash chromatography on silica gel eluting with 3: 2 hexanes: ethyl acetate to give compound 11 (174 mg) as a white solid.

Step 3: Compound 11 (148 mg, 0.33 mmol) was dissolved in 1 N aqueous sodium hydroxide solution (3 ml) and methanol (10 ml) and the resulting solution was refluxed for 1 hour. Most of the methanol was removed in vacuo. Water was added, the mixture was sonicated and filtered. The collected solid was dried under vacuum to give 12 (82 mg) as a white solid. Selected ¹ H NMR data: (CD ₃ OD, 500 MHz) δ 5.48 (d, 1 H, J = 2.8 Hz), 4.04 (d, 1 H, J = 2.7 Hz), 3.74 (dd, J = 8.3 Hz, J = 2.5 Hz), 3.56 (t, 1 H, J = 8.5 Hz), 3.43 (dt, 1 H, J = 7.6 Hz, J = 4.2 Hz), 1.25 (s, 3H), 0.75 (s, 3H). Melting Point: 144-147 ° C.

(VI), 16α-bromoandrost-5-en-3β-ol-11β-acetoxy-17-one (VII), (VIII), androst-5-ene-3β, 11β, 16α-triacet Oxy-17-one (IX), androst-5-ene-3β, 11β, 16α-triacetoxy-17β-ol (X), androast-5-ene-3β, 11β, 16α, 17β-tetrol (XI) .

II. Compound I (4.0 g, 11.4 mmol) was dissolved in 100 ml anhydrous 1,4-dioxane. Sodium methoxide (3.0 g, 55.2 mmol) was added and the mixture was refluxed under anhydrous conditions for 3 hours while monitoring by HPLC. The solvent was removed to one third of the volume and the mixture was acidified to pH = 5-6 with w / 2N HCl. The mixture was extracted with 3 x 50 ml DCM. The organic layers were combined again and washed with 50 ml of saturated sodium bicarbonate and 50 ml brine. After drying over sodium sulphate and evaporation of the solvent, 3.56 g of 95% purity product was isolated.

III. Compound II (13.5 g) and p-toluenesulfonic acid (2.45 g) were stirred for 18 h in 175 ml acetic anhydride anhydride. The mixture was poured into 800 g of ice and stirred for 1 hour. Filtration through a short silica gel plug gave 3.14 g of Compound III .

IV. Compound III (100 mg) was dissolved in 1.55 ml ethylene glycol, then triethyl orthoformate (3.77 ml) and p-toluenesulfonic acid (50 mg) were added. The mixture was refluxed for 1.5 hours and then poured into a hot mixture of 6 ml methanol and 0.08 ml pyridine. The mixture was cooled down and 15 ml of water was added. The mixture was extracted with 3 x 30 ml ethyl acetate and then washed with saturated sodium bicarbonate (20 ml) and brine (20 ml). Drying in sodium sulphate and evaporation of the solvent resulted in 50 mg of compound V in 97% purity.

V. To a 50 mg IV solution in 2 ml DMF was added a 27 mg sodium borohydride solution dissolved in 0.5 ml water at room temperature. The solution was heated to 100 ° C. with vigorous stirring for 15 minutes and then cooled to room temperature. The reaction was poured into 18 ml water and then 0.2 ml acetic acid. The mixture was extracted with ethyl acetate (2 x 10 ml) and then washed with saturated sodium bicarbonate (10 ml) and brine (10 ml). Drying with sodium sulfate and evaporation of the solvent gave 39 mg of compound V.

VI. Compound V (50 mg) and p-toluenesulfonic acid (2 mg) were suspended in a mixture of 2 ml acetone and 0.21 ml water and refluxed for 1.5 hours. After evaporation of acetone, 10 ml water was added and the product precipitated out of solution. Filtration gave 42 mg of desired product in 95% purity.

VII. Compound VI (315 mg) and CuBr ₂ (611 mg) were added to 7 ml anhydrous methanol and refluxed for 24 hours. The reaction mixture was cooled down and then poured into 15 ml hot water and the crude product was filtered off. The crude product was dissolved in 25 ml methanol / THF (1: 1) followed by the addition of 200 mg of activated carbon. This solution was boiled for 10 minutes and then carbon was filtered off. The crude product was recrystallized in methanol to give 426 mg of product in 75% purity.

VIII. Compound VII (420 mg) was dissolved in 18 ml DMF and 7 ml water mixture. An aqueous sodium hydroxide solution was added (1N, 1.31 ml) with stirring. After 10 minutes, the solution was poured into an ice / water mixture containing 1.5 ml 1M HCl. This solution was saturated with NaCl and extracted with 2 × 5 ml ethyl acetate. After drying over sodium sulfate, the solvent was evaporated and the crude product was purified by column chromatography to yield 300 mg of 95% purity Compound VIII.

IX. Compound VIII (300 mg) was dissolved in 6 ml pyridine and 0.34 ml acetyl chloride was added. The reaction was stirred for 18 hours and then poured into 30 ml ice water. The crude product was filtered off and then purified by column to yield 180 mg of pure product.

X. Compound IX (120 mg) was dissolved in 5 ml methanol and cooled in a cold water bath. Sodium borohydride (11.5 mg) was added for 5 minutes and the cold water bath was removed. After 1 hour quenched with 0.2 ml acetic acid and 15 ml water was added. The mixture was extracted with 3 x 20 ml ethyl acetate and washed with saturated sodium bicarbonate (20 ml) and brine (20 ml). After drying over sodium sulphate, column chromatography gave 86 mg of the desired product.

XI. Compound X (180 mg) was dissolved in 5 ml dry ethyl ether and cooled to -78 ° C. Methyl magnesium bromide was dropped (1.2 ml, 1M in ethyl ether). The reaction was warmed to room temperature and refluxed for 3 hours. The reaction was quenched and then neutralized with 1 M HCl. The precipitated product was filtered and then recrystallized three times in methanol / water to yield 36 mg of pure XI. Melting point = 220.3-221.6 ° C. Selected NMR shifts: ¹ H NMR (CD ₃ OD): 5.20 ppm (bs, 1H), 4.26 ppm (dd, J = 3Hz, 5Hz, 1H), 3.88 ppm (m, 1H), 3.32 ppm (m , 1H), 3.19 ppm (d, J = 8 Hz, 1H), 1.24 ppm (s, 3H), 0.92 ppm (s, 3H).

Androst-5-ene-3β, 16α-diacetoxy-7,17-dione (4), androast-5-ene-3β, 16α-diacetoxy-7β, 17β-diol (HE3467).

Synthesis of 2. 1 in THF (100 ml) (3.44 g, 10 mmol) and TMS-Cl (2.15 ml, 16.5 mmol) solution were cooled to −78 ° C., then 2.0 M LDA (7.5 ml, 15 mmol) was added dropwise. The solution was stirred for 30 minutes and warmed up to room temperature. The reaction mixture was partitioned between 100 ml 1: 1 hexanes / ether and 100 ml water. The organic layer was washed with brine (3 x 30 ml) and then dried over Na ₂ S0 ₄ . Yellow oil was formed after the solvent was removed. The crude product was chromatographed on silica gel with 5-20% EtOAc / hexanes to recover 1.9 g of 1 and divalent white solids (600 mg, 1.54 mmol) in 31% yield.

3, synthesis. To a 2 (100 mg, 0.26 mmol) solution in THF (3 ml) cooled to 0 ° C., mCPBA (77%, 62.2 mg, 0.27 mmol) was added and warmed to room temperature. 0.5N HCl (3 ml) was added, stirred for 20 minutes and extracted with ether. The extract was washed with saturated sodium bicarbonate, brine and then dried over Na ₂ SO ₄ . After removal of the solvent, product 3 (90 mg, 0.26 mmol) was produced in 100% yield.

4, synthesis. Acetyl chloride was added dropwise to a solution of 3 (721 mg, 2.0 mmol) in pyridine (10 ml) cooled to 0 ° C. and stirred at 0 ° C. for 2 hours. The reaction was quenched with water (300 ml) and stirred for 15 minutes. A solid formed, which was collected by filtration. The solid was washed with water, 1N HCl and water and dried in vacuo to yield an off-white solid 4 (737 mg) in 90% yield.

Synthesis of HE3467. To a solution of 4 (300 mg, 0.75 mmol) in 1: 1 MeOH / THF (15 ml) cooled to −15 ° C., NaBH ₄ (42.5 mg, 1.12 mmol) was added over 15 minutes. To a solution of cesium chloride (300 mg, 0.81 mmol) in MeOH cooled to −15 ° C. was stirred for 2 minutes. The reaction was quenched with 1 N HCl and poured into 90 ml water. A solid formed, which was collected by filtration. The solid was washed with 1 N HCl and water and dried in vacuo resulting in HE3467 (183 mg, 0.45 mmol) in 60% yield. ¹ HNMR (CD3OD): δ 5.29 (s, 1H), 4.89 (m, 1H), 4.55 (m, 1H), 3.74 (d, 1H, J = 6.52), 3.60 (d, 1H, J = 4.76), 2.36 (d, 2H, J = 1.27), 2.15-2.18 (m, 2H), 2.05 (s, 3H), 2.01 (s, 3H), 1.9-1.1 (m, 11H), 1.11 (s, 3H), 0.82 (s, 3 H).

5α-androstan-2β, 3α, 16α, 17β-tetrol (22)

Step 1: To a solution of 13 (50.0 g, 0.172 mol) in pyridine (150 ml) was added p-toluenesulfonyl chloride (47.0 g, 0.24 mol) at 0 ° C. The reaction mixture was stirred at 0 ° C. for 2 hours and then at room temperature overnight. Water was added. The resulting precipitate was collected by filtration and washed with water. The crude product was purified by recrystallization in methanol to give 14 (75.2 g) of the compound as a white solid.

Step 2: A mixture of compound 14 (75 g, 0.169 mol) in 2.4,6 collidine (200 ml) was refluxed for 5 hours. After quenching, water (500 ml) was added and the resulting precipitate was collected by filtration and washed with water. The solid was recrystallized in methanol to give the crude product (42.5 g). The solid was recrystallized in methanol to give the crude product (42.5 g). The crude product (20.0 g) was dissolved in chloroform (113 ml) and acetic anhydride (37 ml). To this solution, a concentrated sulfuric acid solution (3 ml) and 13 ml acetic anhydride in chloroform (37 ml) were added at 0 ° C. The reaction mixture was stirred at 0 ° C. for 0.5 h, then 700 ml water were added and stirred at rt for 6 h. The resulting precipitate was collected by filtration, washed with water and dried in vacuo, resulting in 15 (17.2 g) as a white solid.

Step 3: A mixture of 15 (8.17 g, 0.030 mol) and copper bromide (10.8 g, 0.046 mol) in anhydrous methanol (220 ml) was refluxed for 18 hours. After quenching, most of the solvent was removed in vacuo and water (150 ml) was added. The resulting precipitate was collected by filtration and washed with water. The solid was recrystallized in methanol, resulting in 16 as a white solid (6.76 g).

4 steps; To a stirred solution of 16 (6.69 g, 0.019 mol) in N, N-dimethylformamide (180 mL) was added 1 N aqueous sodium hydroxide solution (22 ml, 0.022 mol). The reaction mixture was stirred at rt for 0.5 h. 1 N aqueous hydrochloric acid solution (3 ml) and 100 ml water were added. The resulting solution was extracted with ethyl acetate (3 × 250 ml). The combined extracts were dried over magnesium sulphate, filtered and concentrated in vacuo to give 17 (4.37 g) as a waxy solid.

5 steps; To a solution of 17 (3.74 g, 0.013 mol) in pyridine (20 ml) was added acetyl chloride (2.18, 0.028 mmol) at 0 ° C. The reaction mixture was stirred at 0 ° C for 1 h. The resulting mixture was warmed up to room temperature and stirred for 1 h more. Water was added. The precipitate was collected by filtration and washed with water. The solid was dried under vacuum to give 18 (4.25 g) as a white solid.

6 steps; To 18 (2.4 g, 0.0072 mol) solution in methanol (80 ml) sodium borohydride (1.2 g, 0.031 mol) was added at 0 ° C. The reaction mixture was stirred at 0 ° C. for 1 hour. Acetic acid (6 ml) and water (15 ml) were added to quench the mixture. Most of the methanol was removed under reduced pressure. The remaining sludge was partitioned between ethyl acetate (80 ml) and water (20 ml). The organic layer was washed with 1N aqueous hydrochloric acid solution, neutralized with saturated aqueous sodium bicarbonate solution, then dried over magnesium sulfate, filtered and evaporated to afford the crude product 19 (1.92 g) as a white solid.

Step 7: To a solution of 19 (1.9 g, 0.0057 mol) in pyridine (20 ml) was added acetyl chloride (1 ml, 0.014 mol) at 0 ° C. The reaction mixture was stirred at 0 ° C. for 1 hour. The resulting mixture was warmed up to room temperature, stirred for 1 hour more, and then most of the solvent was removed under reduced pressure. The remaining sludge was partitioned between ethyl acetate (80 ml) and water (20 ml). The organic layer was washed with 1N aqueous hydrochloric acid solution, saturated aqueous sodium bicarbonate solution, then dried over magnesium sulfate, filtered and evaporated to give the crude product. The crude product was purified by flash chromatography on silica gel and eluted with 1:10 ethyl acetate: hexanes to give 20 (1.4 g) as a white solid.

Step 8: To a solution of 20 (980 mg, 2.61 mmol) in chloroform (25 ml) m -chloroperoxybenzoic acid (3.6 mmol) was added. The reaction mixture was stirred at rt for 2 h. The organic layer was washed with saturated aqueous sodium bicarbonate solution, washed with water and then dried over magnesium sulfate, filtered and evaporated to give the crude product. The crude product was purified by flash chromatography on silica gel eluting with 1:10 ethyl acetate: hexanes to give 21 (780 mg) as a white waxy solid.

Step 9: A solution of 21 (625 mg, 1.61 mmol) in acetic acid (8 ml) was refluxed for 5 hours. After quenching, the solvent was removed in vacuo, resulting in an oil, which was further dried overnight in vacuo. The resulting waxy solid was dissolved in 2 N aqueous sodium hydroxide solution (8 ml) and methanol (15 ml) and the product was refluxed for 1 hour. Methanol was removed in vacuo and water was added. The resulting precipitate was collected by filtration and washed with water and hot acetone. The collected solid was dried in vacuo resulting in androstane-2β, 3α, 16α, 17β-tetrol or 22 (295 mg) as a white solid. Selected ¹ H NMR data: (CD ₃ OD, 500 MHz) δ 3.98 (d, 1 H, J = 4.8 Hz), 3.79 (br, 1 H), 3.74 (br, 1 H), 3.35 (d, 1 H , 3.6 Hz), 0.99 (s, 3H), 0.77 (s, 3H). mp :. 260-263 ° C.

It is clear that the compounds described herein can also be used to prepare other compounds of Formula 1, for example, other esters or ethers of these compounds. Intermediates during the preparation of the main compounds can also be used in the methods described herein.

Example 23

The ability of the compound of formula 1 to treat multiple sclerosis (MS) was evaluated in experimental autoimmune encephalomyelitis (EAE) in substantially the same manner as described in Example 6 above. Protocols for performing in EAE animal models are described in (D. Auci et. Al., Ann. NY. Acad. Sci. USA , 1051: 730-42, 2005). Female SJL mice (6-8 weeks of age, 25 g average weight) were purchased from Charles-River, kept under standard laboratory conditions with free water and feeding (without nonspecific germs) and surrounding environment prior to starting the study. Adapted for 1 week. The animals were randomly divided into 6 groups of 7 animals in each group, which were as follows: (1) vehicle treated mice, (2) SU5416 (Z-3-[(2,4-dimethyl) Pyrrole-5-yl) methylidenyl] -2-indolinone), (3) mice treated with 17β-ethynylandrost-5-ene-3β, 7β, 17α-triol, (4 ) Mice treated with androst-5-ene-3α, 7β, 16α, 17β-tetrol, (5) mice treated with androst-5-ene-3β, 7β, 16α, 17β-tetrol, (6) 3α -Mice treated with -trifluoromethylandrost-5-ene-3β, 17β-diol, (7) mice treated with 17α-trifluoromethyl-androst-5-ene-3β, 17β-diol, and ( 8) Mice treated with 5α-androstan-3β, 17β-diol-16-oxime. EAE was induced with 200 μl of a 1: 1 emulsion of 75 μg proteolipid protein (PLP) and 6 mg / ml Mycobacterium tuberculosis H37RA in complete Freund's adjuvant (CFA). 200 μl injections were divided into four sites linked to the armpit and inguinal lymph nodes. Pertussis toxin was used as a co-adjuvant, ip injected at 200 ng / mouse on day 0 after immunization and on day 2 post immunization. Treatment with 0.1 mg of compound in 100 μL vehicle, or vehicle alone with qd po (oral feeding), started at the clinical start date of the disease and lasted up to 30 days after immunization. Clinical onset is defined as the time when the clinical symptoms of the disease achieved a grade 2-3 in 25% of mice. The clinical grade was performed by an observer who did not know whether it was treated: 0 = not sick, 1 = limp tail, 2 = moderate paralysis, 3 = severe paralysis, 4 = death status, 5 = death. For significant differences in clinical scores, unpaired data were statistically analyzed by ANOVA and nonparametric Mann-Whitney test. P values <0.05 were considered statistically significant. For statistical analysis, only 5 mice died with EAE on the day of death, which was excluded from the experimental group.

As expected, taxonomic characteristics of EAE were observed in 8 (100%) of 8 mice treated with vehicle within 19 days after immunization. Mean start date was 15.5 ± 3.9 (SD). In the animal group, the duration of disease was 12.3 ± 4.3 days. The mean cumulative score from 1 to 30 days was 24.8 ± 7.8 and the mean cumulative score from 31 to 54 days (post-treatment) was 22.7 ± 15.8. A route of EAE that is very similar to that observed in mice treated with vehicle is SU5416, androst-5-ene-3α, 7β, 16α, 17β-tetrol and 5α-androstan-3β, 17β-diol-16-oxime Observed in animals treated with, the mice so treated showed cumulative disease incidence, disease duration and mean cumulative onset score similar to those of the control group. In contrast, androst-5-ene-3β, 7β, 16α, 17β-tetrol, 3α-trifluoromethylandrost-5-ene-3β, 17β-diol or 17α-trifluoromethylandrost-5 Mice treated with -N-3β, 17β-diol showed a significantly improved pathway of EAE compared to vehicle treated mice, causing a significant reduction in one or more of the mean cumulative scores and duration. On the contrary, none of these three compounds significantly affected the cumulative incidence or lethality of EAE. Finally, 17β-ethynylandrost-5-ene-3β, 7β, 17α-triol showed a trend toward reduced cumulative scores and duration compared to mice treated with vehicle, but the effect was biologically significant. Judged (14.9 ± 17.6 and 7 ± 7.9 vs 24.8 ± 7.8 and 12.3 ± 4.3). The lack of statistical significance for this compound is probably due to the large number of mice assigned zero points during the observation period, resulting in a large standard deviation.

On the last day of treatment on day 30, mice were further monitored up to 24 hours. Chronic disease could be observed in vehicle treated mice having a cumulative score comparable to the duration of treatment. The cumulative score increased substantially during the follow-up period as compared to the treatment period, as the SU5416 with an average cumulative score passed from 25.5 ± 8.9 to 35.5 ± 13.2, more modestly with an average cumulative score. Observed using 17β-ethynylandrost-5-ene-3β, 7β, 17α-triol, passed from 14.9 ± 17.6 to 18.4 ± 20.6. In mice treated with 17β-ethynylandrost-5-ene-3β, 7β, 17α-triol, an increase in EAE incidence of 57.1% at the end of the treatment period to 85.7% at the end of the follow-up period can be observed. there was. On the other hand, other compounds appear to maintain similar cumulative scores in the follow-up period as in the treatment period. This was especially pronounced for 3α-trifluoromethylandrost-5-ene-3β, 17β-diol, which was passed with an average cumulative score of 11.2 ± 4.8 to 10.8 ± 0.3 at the end of the follow up period during the treatment period.

These results are 17β-ethynylandrost-5-ene-3β, 7β, 17α-triol, androst-5-ene-3β, 7β, 16α, 17β-tetrol, 3α-trifluoromethylandrost -5-ene-3β, 17β-diol and 17α-trifluoromethyl-androst-5-ene-3β, 17β-diol show potent anti-inflammatory properties in PLP-derived EAE mouse models in SJL mice Shows. Notable for the interpretation of these findings, particularly for clinical settings, is that compounds were given an EAE model, even when 24% of mice had already shown clinical signs of EAE, even when given in a protocol started 12 days after immunization. Is found to be active. Of particular note is the discovery that the SU5416 was ineffective at this setting. SU5416 has been reported to be effective in EAE (L. Bouerat et al., J. Med. Chem . 48: 5412-5414, 2005). However, to obtain this result, the SU5416 compound was administered simultaneously with immunization of the animal. In contrast, in this protocol, compounds such as 17β-ethynylandrost-5-ene-3β, 7β, 17α-triol were not administered to animals until the symptoms of the disease became evident, which effectively It can be used to treat an already existing disease, or to prevent or delay the onset of the disease.

Example 25

Treatment of ionizing radiation exposure. The effect of the compound of Formula 1 selected on female B6D2F1 mice investigated at lethal levels was compared to control animals treated with vehicle only. Animals were exposed to a total gymnastic dose of 10 Gy at 2.5 Gy / min using a ¹³⁷ Cs source. A group of 12 animals was used for a total of five groups. For groups 1, 2, 3, and 5, test articles were administered continuously for 3 days by subcutaneous injection in a volume of 100 μl and the first dose was administered 2-4 hours after exposure to radiation. For group 4, test articles were administered continuously for 3 days by intramuscular injection in 50 μl volume. The formulation was a suspension containing 0.1% w / v carboxymethyl-cellulose, 0.9% w / v sodium chloride and 0.05% v / v phenol. Prior to injection, the compound of formula 1 was stirred and resuspended constantly and injected into the animals within minutes of placing in the syringe to prevent settling in the syringe.

Groups of animals were treated as follows. Group 1 received only 3 consecutive vehicles by subcutaneous injection daily. Group 2 received 0.6 mg of 3β, 17β-dihydroxyandrost-5-ene in 100 μl of suspension for 3 consecutive days by subcutaneous injection daily. Group 3 injected 3.0 mg of 3β, 17β-dihydroxyandrost-5-ene in a 100 μl suspension for 3 consecutive days by subcutaneous injection daily. Group 4 injected 0.6 mg of 3β, 17β-dihydroxyandrost-5-ene in a 50 μl suspension for 3 consecutive days by intramuscular injection daily. Group 5 received 0.6 mg of 3β-hydroxy-17β-aminoandrost-5-ene in a 100 μl suspension for 3 consecutive days by subcutaneous injection daily. Survival of the animals was observed for 21 days after irradiation, and the following results were obtained. The number of surviving animals is shown for days 6, 7, 12 and 21.

 Days group 6 7 12 21 1 vehicle control 12 11 4 One 2 0.6 mg s.c. 12 11 10 7 3 3.0 mg s.c. 12 12 9 7 4 0.6 mg i.m. 12 12 11 9 5 0.6 mg s.c. 12 12 12 11

Example 25

Treatment of gastrointestinal inflammation. The ability of compounds of Formula 1 to limit or inhibit inflammation or symptoms of inflammation has been demonstrated using animal models for inflammatory bowel disease. Groups of three male Wistar rats (180 ± 20 g) were fasted for 24 hours, followed by 2,4-dinitrobenzene sulfonic acid (DNBS) or saline challenge. 0.5 ml of an ethanol solution of DNBS (30 mg in 0.5 ml of 30% ethanol in saline solution) was instilled into the colon to induce peripheral colitis, followed by injection of 2 ml air through the cannula to maintain the solution in the colon. It was made. The volume used was 0.1 ml per injection of 2 and 20 mg / ml of androst-5-ene-3β, 7β, 17β-triol in liquid formulation, which was administered by subcutaneous injection once daily for 6 days (0.2 mg / animal per day or 2.0 mg / animal per day). The formulation contained 100 mg / ml of the compound in a non-aqueous suspension, for example 2% benzyl alcohol w / v, 0.1% Brij 96 w / v and the same volume of PEG 300 and propylene glycol. Concentrations of 2 mg / mL and 20 mg / mL were obtained as a formulation of a 20 mg / ml formulation containing vehicle lacking compound.

The first dose was 30 minutes after the DNBS challenge. After sulfasalazine (30 mg / mL in 2% Tween 80 in distilled water) once daily (10 mL / kg / day) for 7 consecutive days of oral administration (PO), the first two doses were administered 24 hours prior to DNBS challenge and It was carried out at 2 hours. Examination of the rectum revealed the presence of diarrhea daily. Animals were fasted 24 hours before sacrifice. Animals were sacrificed on day 7 or 8 and their colons were removed and weighed. Prior to colon removal, any signs of adhesion between the colon and other organs were recorded. In addition, after weighing each colon, signs of ulcer were examined. The "net" change in colon to body weight (BW) ratio was normalized to the baseline group challenged with saline. A 25-30% reduction in the "net" colon to weight ratio was considered significant. The results indicate that androst-5-ene-3β, 7β, 17β-triol has a moderate effect on disease progression (about 15-20% reduction in net colon-to-weight ratio), but 17α-ethynylandrost -5-ene-3β, 7β, 17β-triol or androast-5-ene-3β, 7β, 16α, 17β-tetrol have been shown to be effective (a reduction of about 25-35% in the net colon to weight ratio) .

Modifications to this protocol include administration of one or more of the above-described dosage levels and / or 0.05 mg / animal per day, 0.1 mg / animal per day, 0.5 mg / animal per day and 1.0 mg / animal per day Dosage levels are used to administer the compound in an aqueous solution of 30% sulfobutylether-cyclodextrin in water.

Example 26

Treatment of neuronal loss associated with trauma and osteoporosis or bone loss symptoms. Immune competence is a complex function in which endogenous glucocorticoid (GC) levels can be acutely deleted after an increase in trauma. Compound 5-androsten-3β, 7β, 17β-triol is used to preserve immune function by retaining trophic or assimilation. In animal models of acute cerebral ischemic stroke associated with occlusion of bilateral carotid arteries in gerbils, treatment with 5-androsten-3β, 7β, 17β-triol significantly improved cognitive abilities compared to stroke alone ( p = 0.03). Therefore, the time delay for finding the measured food in each group was 6.9 ± 0.9 seconds (sec) for sham, 46.9 ± 13.6 seconds for stroke alone, and 5-androsten-3β, 7β, 17β- 14.8 ± 4.8 seconds for stroke treated with triol. Incidentally, the stroke-induced loss of CA1 hippocampal neurons was significantly recovered by treatment with 5-androsten-3β, 7β, 17β-triol (Sham = 362,247 ± 6,839; stroke = 152,354 ± 11,575; And stroke + 5-androsten-3β, 7β, 17β-triol = 207,854 ± 47,334).

In the symptoms of bone loss, 5-androsten-3β, 7β, 17β-triol influenced the basic bone structure, namely the cortical and trabecular and growth plates. Loss of cortical (femoral) and columnar / cavernous (tibia) bone mass when treated with thermal trauma (20% of body surface area) with 5-androsten-3β, 7β, 17β-triol , And inhibition of chondrocyte proliferation in the proximal tibial epiphyseal growth plate was significantly (p <0.01) prevented by 5-androsten-3β, 7β, 17β-triol treatment. Histological analysis of the femoral cortical bones supported the increased rate of bone formation. We also observed partial protection against loss of bone mineral content observed through double X-ray absorptiometry. The femoral ash weight was significantly (p <0.01) larger than in vehicle treated mice, indicating that 5-androsten-3β, 7β, 17β-triol preserved the bone mineral content. The pre-inflammatory effects of chronically high GC levels in the brain support that elevated GC levels exacerbate the consequences of neurological injury. Upstream metabolic precursors of the adrenal steroids DHEA (5-androsten-3β-hydroxy-17-one), 5-androsten-3β, 7β, 17β-triol have been demonstrated to prevent dexamethasone-induced thymic degeneration in mice (KL Blauer et al., Endocrinology , 129: 3174, 1991). Taken together, these results indicate that 5-androsten-3β, 7β, 17β-triol inhibits GC induced loss of functional neural tissue and preserves bone structure after burn due to heat.

5-androsten-3β, 7β, 17β-triol, 17α-ethynyl-5-androsten-3β, 7β, 17β-triol and 4-estrenen-3α, on the adverse effects of glucocorticoids on bone growth The ability of compounds comprising 17β-diol was observed in human MG-63 osteosarcoma cell line. MG-63 cells are osteoblasts, cells that mediate bone growth. These cell lines are used intensively to study bone biology and to analyze the biological activity of the compounds for the treatment of bone loss symptoms (eg, BD Boyan et al., J. Biol. Chem ., 264 (20): 11879-11886, 1989; LC Hofbauer et al., Endocrinology , 140 (10): 4382-4389, 1999). Toxicity, a side effect associated with elevated glucocorticoid levels, includes decreased production of IL-6 and IL-8 by osteoblasts, including MG-63 cell lines, and the 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD Increased expression). Increased 11β-hydroxysteroid dehydrogenase type 1 enzyme increases endogenous glucocorticoid activity as endogenous cortizone converts to active cortisol, which in turn inhibits bone growth. 11β-HSD enzyme is expressed in liver, fat cells, brain and bone tissue. Cortisol produced by 11β-HSD-1 contributes to central nervous system diseases such as osteoporosis, insulin resistance, type 2 diabetes, dyslipidemia, obesity, stroke, neuropathy, depression and Parkinson's disease. Reduction of IL-6, IL-8 and osteoprotegerin are associated with a decrease in bone growth by osteoblasts. Pilot studies showed that the IC ₅₀ for the inhibition of IL-6 of MG-63 cells by dexamethasone was 10 nM and the IC ₅₀ for the inhibition of growth of MG-63 cells by dexamethasone was 15.3 nM.

In this protocol, MG-63 cells were grown in the presence or absence of synthetic glucocorticoid dexamethasone at 30 nM concentration and in the presence or absence of the compound of formula 1 at 10 nM. Compound 1 in the table below was 5-androsten-3β, 7β, 17β-triol, compound 2 was 17α-ethynyl-5-androsten-3β, 7β, 17β-triol, and compound 3 was 4-ester Nene-3α, 17β-diol. The results for these compounds are shown below.

MG-63 growth conditions IL-6 pg / ml IL-8 unit 11β-HSD mRNA unit Bone resorption inhibitor pmol / l Vehicle control 6.2 0.90 0.25 445 Dexamethasone 1.3 0.12 1.0 280 Compound 1 4.0 0.53 0.73 --- Compound 2 2.8 0.50 0.54 --- Compound 2 (nM) --- --- --- 455 Compound 3 4.1 0.55 0.75 ---

These results show that 10 nM of compounds partially inhibited the side effects of 30 nM of dexamethasone, which is also associated with elevated levels of glucocorticoids in osteoblasts in cells that mediate bone growth. It also shows that it can inhibit the toxicity. In a related protocol, 1 nM of 17α-ethynyl-5-androsten-3β, 7β, 17β-triol was also grown after 7 hours in the presence of 30 nM dexamethasone, MG, as shown in the table above. Completely inhibits the synthesis of bone resorption inhibitors following -63 cells. Bone repressor is a factor associated with bone growth, and reduced bone resorption factor synthesis is associated with bone loss. Another compound that completely or partially inhibits the decrease in bone resorption inhibitor synthesis by MG-63 cells in the presence of 30 nM dexamethasone is 17α-trifluoromethyl-5-androsten-3β, 7β, 17β-triol (dexamethasone Normal or reference bone resorption factor at 1 μM level of compound compared to vehicle control without), 5-androsten-3β, 7β, 16α, 17β-triol (normal bone resorption factor level of 0.1 μM) , 3β, 7α, 16α, 17β-tetrahydroxyandrost-5-ene (level of bone resorption inhibitor near normal at 10 nM), 3α, 7β, 16α, 17β-tetrahydroxyandrost-5-ene ( Normal bone resorption factor levels at 10 nM), 17α-methylandrost-5-ene-3β, 17β-diol-7-one (increased bone resorption factor levels at 100 nM), 17α-methylandrost-5 -N-3β, 7β, 17β-diol (normal bone resorption inhibitor levels at 10 nM). Another compound that partially inhibits the reduction of bone resorption inhibitors in the presence of 30 nM dexamethasone was androst-5-ene-3β, 17β-diol-7-oxime.

In a similar protocol, compound 3α, 17β-dihydroxyandrost-4-ene prevents dexamethasone induced inhibition of IL-8 and IL-6 and reduction of dexamethasone induced 11β-HSD mRNA by MG-63 cells. Statistically significant level of inhibition.

To demonstrate that the corresponding effect can be obtained in vivo, compound 17α-ethynyl-5-androsten-3β, 7β, 17β-triol is administered to mice daily with dexamethasone for 23 days, in animals. Lowered bone resorption factor. Bone uptake inhibitor levels in mice treated with vehicle and 10 μg / 1 day (positive control) were 3.3 pmol / L bone uptake inhibitors, but vehicle, dexamethasone and 17α-ethynyl-5-androsten-3β, 7β, The bone resorption factor of mice administered 4 mg / kg / day with 17β-triol was 6.4 pmol / L (p <0.05).

The degree of apoptosis of osteoblasts and osteocytes in bones of murine and vertebrate animals was examined as a function of estrogen deficiency. Ovaries of Swiss Webster mice (4 months old) were removed. After 20 days, the animals were sacrificed and the spine were separated, fixed and embedded and then decalcified in methacrylate. The spread of osteoblasts and osteocytic apoptosis was examined by the TUNEL method using CuSO ₄ enhancement, indicating that estrogen loss was reduced. Mice treated with reference compounds such as 17β-estradiol and compounds of formula 1 such as 4-estrene-3α, 17β-diol or 17α-ethynyl-5-androsten-3β, 7β, 17β-triol were cells Death was shown to be reduced, consistent with reduced bone loss.

Taken together, the results described in this example show that compounds such as 17α-ethynyl-5-androsten-3β, 7β, 17β-triol affect bone tissue by increasing bone growth and inhibiting bone loss. It's proof that you're crazy. Compounds such as 17α-ethynyl-5-androsten-3β, 7β, 17β-triol and 5-androsten-3β, 7β, 16α, 17β-tetrol are androgen receptor, estrogen receptor-α, or estrogen receptor- It does not interact with β, which is consistent with their ability to treat bone loss symptoms without exerting undesirable sex hormone activity.

Example 28

A thermal imaging model using mouse ear tissue was used to characterize the ability of compounds to treat inflammation associated with thermal trauma. The above symptom was a minimal burn that progressed to tissue necrosis in the exposed ear of untreated mice 24 to 72 hours after the burn. In the group of Balb / c mice, approximately 9 week old mice were labeled and divided into control and treatment groups. After recording the thickness of the ear soaked in hot water, the whole ear of the anesthetized mouse was soaked in 52 ° C. water for 24 seconds. Each mouse was returned to the cage after injecting 100 mg of the compound in propylene glycol (control) or propylene glycol. The degree of swelling of the ears was observed for each mouse at various time points before and after the image. The degree of swelling was observed in each mouse before and 1, 3, 6, 9, 12, 18, 24 and 48 hours after burn. Animals were treated with 100 mg of dehydroepiandrosterone (DHEA) dissolved in propylene glycol. Analysis of edema formation and disappearance in the control and DHEA-treated mice revealed that the earliest swelling resulted in the degree of edema in mice treated with DHEA and untreated burns 6 hours after trauma. Turned out to be.

In the untreated control, the degree of edema began to decrease within 12 hours, and then the rate of edema reduction rapidly increased over the next 12 hours. Between 24 and 48 hours after the burn, the distribution of the ear tissue appeared to be lost more than the original microvascular distribution. Compounds androst-5-ene-3β, 17β-diol and 16α-bromodehydroepiandrosterone protected the ischemia from thermal burns in the ears of treated animals. Compound 16α-hydroxydehydroepiandrosterone had a low degree of protection, that is, reduced but not completely prevented progressive ischemia, while 16α-chlorodehydroepiandrosterone only slightly protected against progressive ischemia. gave.

The effect of the compound on hemorrhagic shock and ischemia was examined in another protocol. CF-1 mice 6-8 months of age were anesthetized with methoxyfluorotane and prepared for abdominal surgery. Each mouse was tested for the degree of response to breathing, blinking and skin stings to ensure proper anesthesia for surgery. The duration of the abdominal surgery took approximately 2 hours, with 35-40% of the animal's blood volume removed over 30 minutes. Removing blood in a controlled manner has an effect similar to hemorrhagic shock. The removed blood and 2 × volume of resuscitation solution (lactated Ringers solution) were slowly injected through the central vein. The resuscitation solution was supplemented with excipients as 2 mg dehydroepiandrosterone-3β-sulfate or placebo. The peritoneum and the skin covering it were sewn separately. Animals were left between 38 ° C and 39 ° C until recovery was complete. Under these conditions, animals treated with placebo died within 24 to 48 hours. Four hours after surgery, colony forming units (CFUs) for bacteria were counted and malondialdehyde in the liver was analyzed using conventional techniques. Mesenteric lymph nodes (MLN) were removed, cultured in blood agar plates, and CFUs were counted after incubation. The liver was removed and the amount of malondialdehyde was measured. Treatment with dehydroepiandrosterone-3β-sulfate survived 15 out of 15, but only 1 out of 15 vehicle control animals survived.

The effect on treatment was examined in a rat model of hemorrhagic trauma. Twenty-four rats developed a 40% loss of total blood volume, which consisted of catheterization and laparotomy (soft tissue trauma) to mimic trauma and bleeding. One hour after the onset of bleeding, animals were resuscitated using crystalloid liquid and packed red blood cells (PRBCs). Twelve animals received an hour after the onset of bleeding at a concentration of 40 mL / kg at 100 μl / kg body weight of androst-5-ene-3β, 7β, 17β-triol in a methyl cellulose suspension, but with liquid Subcutaneous injections were made before injection and resuscitation. Twelve animals were injected subcutaneously with a methyl cellulose control at 100 μl / kg body weight. Three days after induction of bleeding, 12 animals receiving androst-5-en-3β, 7β, 17β-triol showed 100% survival, but mortality in the untreated group was 25% (P <0.04). , Barnard's unconditional test using the difference in the two binomial propotion.

A blood pressure reducing hemorrhagic trauma protocol was also performed as the second model of hemorrhagic trauma. In this protocol, 15 rats were bleeding as described above to have an average arterial pressure of about 35-40 mmHg and resuspended 1 hour after the start of bleeding using crystalloid and PRBC. 7 Animals One animal received 40 times of 100 μl / kg body weight of androst-5-ene-3β, 7β, 17β-triol in cellulose suspension, one hour after initiating bleeding, before injecting the resuscitation liquid. Subcutaneous injections were made at a concentration of mg / kg body weight. Eight animals were injected subcutaneously with a methyl cellulose control at 100 μl / kg. Two days after bleeding induction, the mortality rate of the untreated control (n = 8) was 75%. The mortality rate of animals treated with androst-5-ene-3β, 7β, 17β-triol was 43%, demonstrating that the compound is protective in case of hemorrhagic trauma in which blood pressure is reduced.

Example 29

Metabolic stability. Metabolic stability of selected compounds was examined in vitro using microsomes obtained from liver tissue according to the following protocol. The microsomes of this protocol are capable of hydroxylation and redox reactions that interconvert between hydroxyl and ketones on steroid molecules. Microsomes do not mediate conjugation reactions, such as sulfonylation of 3β-hydroxyl groups or glucuronidation between 3α-hydroxyl groups.

The protocol was performed as follows. (1) Prepare 0.5 mM compound in acetonitrile / water 35:65. For androst-5-ene-3β, 17β-diol, 0.145 mg / mL or 1 mg / mL stock is prepared with 29.0 μL + 171 μL solvent. For standard curves, dilutions of 0.5 mM stocks were used to obtain final concentrations of androst-5-ene-3β, 17β-diol at 10 μM, 5 μM and 1 μM. (2) A sample was prepared as follows. Each assay consists of an androst-5-ene-3β, 17β-diol control and 1 to 8 unknown compounds. Tubes for each compound are as follows: 1-0 '2-0' 3-0 '4-0' ^* 5-0 ' ^* 6-5 μM 7-1 μM 8-30' 9-30 '10- 30 '(wherein asterisk (*) indicates denatured microsome negative control reaction tubes. The numbering of additional compounds began with 11, 21, 31, etc. (3) Each tube contained 315 μL PBS (pH 7.3). (7.5) Internal standard / acetonitrile solution (5) NADPH recurrence system (NRS) was 125 μl per tube 1.7 mg / in PBS ml NADP, 7.8 mg / ml glucose-6-phosphate, 6 units / mL glucose-6-phosphate dehydrogenase were added and stored on ice until fresh NRS was used for each experiment. 125 μl of NRS was used for each tube (7) The liver microsome preparations removed in the -80 ° C. freezer were stored and thawed in a water bath at room temperature. The concentration of water was 20 mg / ml Each reaction was diluted to 5 mg / ml concentration in PBS (ie 4 dilutions) using 0.25 mg / tube and then stored on ice. 500 μl acetonitrile of denatured microsome control tube was added at −20 ° C. The 0 o'clock tube was transferred to ice and the denatured microsome control was preincubated for 5 minutes at 37 ° C. (9) Microsome preparations The containing assay tube was preincubated for 5 minutes at 37 ° C. (10) For each incubation tube, the reaction was started by adding 50 μl of the microsome preparation and vortexing the mixture. Termination was achieved by addition and vortexing μl acetonitrile at −20 ° C. (12) After the reaction was completed, 100 μl from each reaction tube was transferred to a new tube and 200 μl of water and 1400 μl of methyl- t -butyl ether Was added to each tube The tubes were vortexed and centrifuged in microfuge for 10 minutes at 13000 rpm. The tube was placed on a dry ice-methanol bath until the aqueous layer became a frozen solid. (13) Methyl- t -butyl ether was transferred from each tube to a new tube, the solvent was ether evaporated under nitrogen and the precipitate was resuspended in 100 μl of acetonitrile / water 35:65 and analyzed by LCMS. The results are shown in the table below based on the incubation times presented.

compound parent remaining human microsome parent remaining mouse microsome Androst-5-ene-3β, 17β-diol 39% (10 minutes) 25% (10 minutes) Androst-5-ene-3β, 17β-diol 30% (90 minutes) --- Androst-5-ene-3β, 7β, 17β-triol 86% (90 minutes) 89% (10 minutes) 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol --- 86% * (30 minutes) Androst-5-ene-3β, 7β, 16α, 17β-tetrol 100% (10 minutes) 100% (10 minutes) Androst-5-ene-3α, 7β, 16α, 17β-tetrol 100% (10 minutes) 100% (10 minutes) Androst-5-ene-3α, 7α, 16α, 17β-tetrol 100% (10 minutes) 100% (10 minutes) Androst-5-ene-3β, 7α, 16α, 17β-tetrol 100% (10 minutes) 100% (10 minutes)  Rat microsomes instead of mouse preparations

The results indicate that the tetrol compound is resistant to redox reactions, which is consistent with significantly reducing the degree of metabolism compared to the androst-5-ene-3β, 17β-diol reference compound. This observation was unexpected because each of the four hydroxyl groups did not reduce the ketone, but was actually affected. Other compounds tested include androst-5-en-3β, 16α, 17β-triol, androstan-3β, 16α-diol-17-one and androstan-3α, 16α, 17α-triol, the compounds All were metabolized by microsomes at a similar rate as the androst-5-ene-3β, 17β-diol reference compound.

Example 30

Drug Uptake Measurement with CaCo-2 Cells. This protocol was used to measure the influx of compounds through CaCo-2 cell monolayers. CaCo-2 cells are human cells with polarized, highly differentiated cell lines that exhibit a visceral uptake cell phenotype (J. Hunter et al., J. Biol. Chem ., 268 (20): 14991-14997, 1993). . This cell line is used to study the rate at which various compounds pass through the cell monolayer. Confluent monolayers of Caco-2 cells were commonly used to model visceral epithelial cells and to obtain permeation coefficients from the state-state flux of test compounds. This can usually provide information as to whether the compound is administered orally and biologically usable.

In this protocol, cells were maintained in medium at 37 ° C. using 100 μl per well of warm medium in sterile 50 ml tubes. Cells were grown on sterile 24 well plates with 600 μl of differentiation medium per well. Wells contained transwell inserts in two compartments per well. 100 μl of differentiation medium was carefully added to each well with the pipette tip touching the well side. Cells were incubated with 37 ° C., 5% CO ₂ and saturated for 48 hours to form a single layer. For each plate, tubes were numbered tubes 1-24 for 'basolateral' buffer to act as basolateral 0 o'clock (T ₀ ). Tubes 26 to 49 was 'apical' buffer containing the test item, and acts as a apical T _o. Tube 51-74 was at T ₂₀ (20 minutes), 76-99 was at T ₄₀ , 101-124 was at T ₈₀ , 126-149 was at T ₁₂₀ , and 151-174 was at mass balance. T ₁₂₀ apical sample for measurement. Tubes 175-179 were the 5-point standard curve for compound 1, tubes 180-184 were the standard curve for compound 2, and tubes 230-234 were the standard curve for compound 12. Tubes 1-49 were laid out in four rows from rack 1, 51-99 in rack 2, 101-149 in rack 3, 151-174 in rack 4, and 175-234 in racks 5 and 6 Deployed.

The buffer was prepared by removing 150 ml of transfer buffer from a fresh 1000 ml bottle (pH 7.4 with 1N HCl). This buffer was 'basolateral'. The remaining pH of 850 mL was adjusted to 6.5 with 1 N HCl for apical buffer. 150 mL of apical buffer was placed in a separate container and the remaining 700 mL was used for washing. The buffer was stored at 4 ° C. and used at room temperature for the protocol.

When the differentiation medium reached room temperature, about 20 ml were poured into a small beaker. Probes were equilibrated in this medium for 15 minutes. 24 wells were removed from the incubator and allowed to reach room temperature. Insert the probe into the well while keeping each well in contact with the cell monolayer; Press the TEST button when the probe is near the medium surface, and read the number from 0000 when the probe touches the surface; Measurements were made by probes using a reading of> 1000 Hz. Apical buffer was removed from the transwell inserts and the entire plate was rinsed in a 1000 ml beaker containing the wash buffer to remove all differentiation buffer. Transwells were transferred to T20 plates. 10 μM of the test compound and 10 μM of the control group (carabamazapine MW 236; hydrochlorothiazide MW 351) (eg 29 μl of 1 mg / ml androst-5-ene-3β, 17β-diol reference solution) By addition to ml of apical buffer, it was added to apical buffer. 0.6 ml of basolateral buffer was added to all wells.

As an internal standard a solution of 50 μg / ml 3α, 7β, 16α, 17β-tetrahydroxyandrost-5-ene, 150 μl of compound (1 mg / ml in ethanol) was added 10 ml of acetonitrile / water (25 : 75) to prepare. Standard curves were prepared in basolateral buffer for each compound. 10 μM apical buffer was diluted 6-fold when delivered to the basolateral compartments, allowing the standard curve to be made at 6-fold lower concentrations.

density Apical TA (10 μM)  Baso buffer 2 μM 120 480 1 μM 60 540 0.5 μM 30 570 0.2 μM 12 588 0.05 3 597

600 μl of basolateral buffer was contained in 1 to 24 tubes for the T _o control. Of 100 ㎕ apical buffer + + Test Items 500 ml apical buffer (Thus, the concentration range of the standard curve) was added to the tubes 26 to 49, and act as an apical T _o. 100 μl apical buffer + compound was placed in the apical portion. The time the transwell was placed on the plate was taken at 0 o'clock (T _o ). At T = 20, transwells were transferred to T40 plates and 600 μl of sample from T20 plates was added to appropriate tubes. At T = 40, transwells were transferred to T80 plates and 600 μl of sample was taken from T40 plates and placed in appropriate tubes. At T = 80, transwells were transferred to T120 plates. 600 μl of sample from the T80 plate was pipetted into a suitable tube and so on for the remaining timepoint tubes. 100 μl of apical buffer was added to the appropriate tube for mass balance. Immediately extracted samples were placed in the freezer.

300 μl of each sample was transferred to a 2 ml tube labeled from the assay tube, but not for tubes 151-174 (which contained only 100 μl); 50 μl of these samples were transferred to 250 μl basolateral buffer and added (Made a 6-fold dilution). 20 μl of 3α, 7β, 16α, 17β-tetrahydroxyandrost-5-ene internal standard was added to each tube and 1500 μl of methyl- t -butyl ether was added to each tube. The tubes were vortexed, centrifuged in a macrofuge for 10 minutes and then placed in a methanol / dry ice bath prior to freezing. New tubes were labeled and methyl- t -butyl ether was transferred from each freezing tube to a new tube. Methyl- t -butyl ether was evaporated under nitrogen, reconstituted in 120 μl acetonitrile / water (35:65) and analyzed by reconstituted LCMS. In the table below, compound 1 is 3β, 7β, 16α, 17β-tetrahydroxyandrost-5-ene, compound 2 is 17α-ethynylandrost-5-3β, 7β, 17β-triol, compound 3 Is 3α, 17β-dihydroxy-17α-ethynylandrostane, compound 4 is 3α, 7β, 16α, 17β-tetrahydroxyandrost-5-ene, and compound 5 is 2β, 3α, 16α, 17β- Tetrahydroxyandrostan, compound 6 is 3β, 16α-diacetoxy-7β, 17β-dihydroxyandrost-5-ene, compound 7 is 3β-acetoxy-17α-ethynylandrost-5- 7β, 17β-diol, compound 8 was 3β-acetoxyandrost-5-7β, 16α, 17β-triol and compound 9 was 17α-ethynylandrost-5-3α, 7β, 17β-triol.

compound 80 min cumulative basolateral concentration (μM) 80 min cumulative basolateral concentration (μM) % Passed to apical for 80 minutes % Passed One 2.195 0.017 0.008 0.8% 2 1.911 0.470 0.246 24.6% 3 2.727 0.411 0.151 15.1% 4 1.664 0.019 0.012 1.2% 5 1.817 0.162 0.089 8.9% 6 1.776 0.185 0.104 17.8% 7 1.710 0.195 0.114 31.4% 8 1.724 0.123 0.071 15.9% 9 1.773 0.531 0.299 29.9%

Studies with CaCo-2 cell lines showed that tetrol compounds such as androst-5-ene-3β, 7β, 16α, 17β-tetrol were not very permeable and therefore expected to be orally active. Not shown. In spite of this, the compound androst-5-ene-3β, 7β, 16α, 17β-tetrol was active when administered orally as described above in mice in a diabetic treatment model. Other protocols include the degree of sulphation for tetro compounds such as 3β, 7β, 16α, 17β-tetrahydroxyandrost-5-ene and 3α, 7β, 16α, 17β-tetrahydroxyandrost-5-ene, and The degree of glucuronization was shown to be lower for the tetrol compound compared to the diol. Such activity may be due at least in part to the low metabolism of the tetrol compound in vivo.

Claims (14)

A composition for preventing or treating symptoms of type 2 diabetes, hyperglycemia, rheumatoid arthritis, osteoarthritis, multiple sclerosis or bone loss, comprising a compound of the following structural formula and at least one excipient:

Wherein ^one of R ¹ is —H or C _1-8 optionally substituted alkyl and the other R ¹ is —OH, C _2-8 ester or C _1-8 ether; One of R ² is —H or C _1-8 optionally substituted alkyl and the other R ² is —H, —OH, C _2-8 ester or C _1-8 ether, or two R ² is Together = NOH; One of R ³ is —H or C _1-8 optionally substituted alkyl and the other R ³ is —H, —OH, C _2-8 ester, C _1-8 ether or C _1-8 Substituted alkyl, or all R ³ together represent = NOH; One of R ⁴ is —H or C _1-8 optionally substituted alkyl and the other R ⁴ is —OH, C _2-8 ester or C _1-8 ether; R ⁵ is —CH ₃ , —C ₂ H ₅ or —CH ₂ OH; R ⁶ is —H, —CH ₃ , —C ₂ H ₅ or —CH ₂ OH; One of R ⁷ is —H or C _1-8 optionally substituted alkyl and the other R ⁷ is —H, —OH, a C _2-8 ester or a C _1-8 ether; R ⁹ is —O— or —C (R ¹² ) (R ¹² ) — wherein one of R ¹² is —H, —F, —Br or C _1-8 optionally substituted alkyl and the other R ¹² is —H, —OH, a C _2-20 ester or a C _1-20 ether or C _1-8 optionally substituted alkyl, or two R ¹² together represent = O, = NOH or = NOCH ₃ ; R ¹⁰ is -H or -F; One of R ¹¹ is —H or C _1-8 optionally substituted alkyl and the other R ¹¹ is —H, —OH, C _2-8 ester, C _1-8 ether or C _1-8 As substituted alkyl, Wherein (1) at least one of R ² is —OH, C _2-8 ester or C _1-8 ether, and one of R ⁷ , R ¹¹ and R ¹² is independently —OH, C _2-8 ester or C _1-8 ether, (2) one of R ⁴ is C _1-8 substituted alkyl or C _2-8 optionally substituted alkyl and is selected from R ² , R ³ , R ⁷ , R ¹¹ and R ¹² At least one is independently —OH, a C _2-8 ester or a C _1-8 ether or (3) at least two of R ² , R ³ , R ⁷ , R ¹¹ and R ¹² are independently —OH, C _{2 -8} ester or C _1-8 ether. The composition of claim 1, wherein the compound has the structure

Wherein ^one of R ¹ is —H or C _1-4 optionally substituted alkyl and the other R ¹ is —OH, —OC (O) CH ₃ , —OC (O) CH ₂ CH ₃ or — OCH ₃ ; One of R ² is —H or C _1-4 optionally substituted alkyl and the other R ² is —OH, —OC (O) CH ₃ , —OC (O) CH ₂ CH ₃ or —OCH ₃ ; ; One of R ³ is —H or C _1-4 optionally substituted alkyl and the other R ³ is —OH, —OC (O) CH ₃ , —OC (O) CH ₂ CH ₃ or —OCH ₃ ; ; One of R ⁴ is —H or C _1-4 optionally substituted alkyl and the other R ⁴ is —OH, —OC (O) CH ₃ or —OC (O) CH ₂ CH ₃ . The composition of claim 1 for the prevention or treatment of type 2 diabetes. The compound of claim 3, wherein the compound is 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androast-5-ene-3β, 7β, 16α, 17β-tetrol, 17α- Tinylandrost-5-ene-3β, 7β, 16α, 17β-tetrol, androst-5-ene-3β, 7α, 16α, 17β-tetrol, androast-5-ene-3β, 4β, 16α, 17β-tetrol, androast-5-ene-3α, 4β, 16α, 17β-tetrol, androast-5-ene-3β, 11β, 16α, 17β-tetrol, androst-5-ene-3α, 11β, 16α, 17β-tetrol, 3β, 11β, 16β, 17β-tetrol, androst-5-ene-3α, 11β, 16β, 17β-tetrol, androst-5-ene-2β, 3α, 16α , 17β-tetrol or a C _2-4 monoester or C _2-4 diester analog of these compounds, optionally said monoester is acetate in the 3- or 17-position, or said diester is 3- and 17- The composition is acetate in position. The composition of claim 1 for the prevention or treatment of multiple sclerosis. The compound of claim 5, wherein the compound is 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androast-5-ene-3β, 7β, 16α, 17β-tetrol, 17α- Tinylandrost-5-ene-3β, 7β, 16α, 17β-tetrol, androst-5-ene-3β, 7α, 16α, 17β-tetrol, androast-5-ene-3β, 4β, 16α, 17β-tetrol, androast-5-ene-3α, 4β, 16α, 17β-tetrol, androast-5-ene-3β, 11β, 16α, 17β-tetrol, androst-5-ene-3α, 11β, 16α, 17β-tetrol, 3β, 11β, 16β, 17β-tetrol, androst-5-ene-3α, 11β, 16β, 17β-tetrol, androst-5-ene-2β, 3α, 16α , 17β-tetrol or a C _2-4 monoester or C _2-4 diester analog of these compounds, optionally said monoester is acetate in the 3- or 17-position, or said diester is 3- and 17- The composition is acetate in the position. The composition of claim 1 for the prevention or treatment of hyperglycemia. The method of claim 7, wherein the compound is 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androst-5-ene-3β, 7β, 16α, 17β-tetrol, 17α- Tinylandrost-5-ene-3β, 7β, 16α, 17β-tetrol, androast-5-ene-3β, 7α, 16α, 17β-tetrol, androast-5-ene-3β, 4β, 16α, 17β-tetrol, androast-5-ene-3α, 4β, 16α, 17β-tetrol, androast-5-ene-3β, 11β, 16α, 17β-tetrol, androst-5-ene-3α, 11β, 16α, 17β-tetrol, 3β, 11β, 16β, 17β-tetrol, androst-5-ene-3α, 11β, 16β, 17β-tetrol, androst-5-ene-2β, 3α, 16α , 17β-tetrol or a C _2-4 monoester or C _2-4 diester analog of these compounds, optionally said monoester is acetate in the 3- or 17-position, or said diester is 3- and 17- The composition is acetate in the position. The composition of claim 1 for the prevention or treatment of osteoarthritis. The composition of claim 1 for the prevention or treatment of rheumatoid arthritis. The compound of claim 10, wherein the compound is 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androast-5-ene-3β, 7β, 16α, 17β-tetrol, 17α- Tinylandrost-5-ene-3β, 7β, 16α, 17β-tetrol, androst-5-ene-3β, 7α, 16α, 17β-tetrol, androast-5-ene-3β, 4β, 16α, 17β-tetrol, androast-5-ene-3α, 4β, 16α, 17β-tetrol, androast-5-ene-2β, 3α, 16α, 17β-tetrol or C _2-4 monoesters of these compounds Or a C _2-4 diester analog, optionally (a) the C _2-4 monoester is acetate in the 3- or 17-position or (b) the C _2-4 diester is the 3- and 17-position In acetate. The composition of claim 1 for preventing or treating bone loss symptoms. 13. The composition of claim 12, wherein the bone loss condition is osteoporosis, fracture, thermal burn, radiation burn or chemical burn or elevated natural glucocorticoid levels or the presence of synthetic glucocorticoids, optionally dexamethasone. The method of claim 13, wherein the bone loss symptoms are osteoporosis, the compound is 17α-ethynylandrost-5-ene-3β, 7β, 17β-triol, androast-5-ene-3β, 7β, 16α, 17β-tetrol, 17α-ethynylandrost-5-ene-3β, 7β, 16α, 17β-tetrol, androst-5-ene-3β, 7α, 16α, 17β-tetrol, androst-5- N-3β, 4β, 16α, 17β-tetrol, androst-5-en-3α, 4β, 16α, 17β-tetrol, androst-5-ene-2β, 3α, 16α, 17β-tetrol or two C _2-4 monoester or C _2-4 diester analogues of the compounds, optionally (a) the C _2-4 monoester is acetate in the 3- or 17-position, or (b) the C _2-4 di Wherein the ester is acetate in the 3- and 17-positions.